The Sbi Protein Contributes to Staphylococcus aureus Inflammatory Response during Systemic Infection

Staphylococcus aureus is an important human pathogen that causes infections that may present high morbidity and mortality. Among its many virulence factors protein A (SpA) and Staphylococcal binding immunoglobulin protein (Sbi) bind the Fc portion of IgG interfering with opsonophagocytosis. We have previously demonstrated that SpA interacts with the TNF-α receptor (TNFR) 1 through each of the five IgG binding domains and induces the production of pro-inflammatory cytokines and chemokines. The IgG binding domains of Sbi are homologous to those of SpA, which allow us to hypothesize that Sbi might also have a role in the inflammatory response induced by S. aureus. We demonstrate that Sbi is a novel factor that participates in the induction of the inflammatory response during staphylococcal infections via TNFR1 and EGFR mediated signaling as well as downstream MAPKs. The expression of Sbi significantly contributed to IL-6 production and modulated CXCL-1 expression as well as neutrophil recruitment to the site of infection, thus demonstrating for the first time its relevance as a pro-inflammatory staphylococcal antigen in an in vivo model.     

Double-stranded RNA induces shedding of the 34-kDa soluble TNFR1 from human airway epithelial cells via TLR3-TRIF-RIP1-dependent signaling: roles for dual oxidase 2- and caspase-dependent pathways

TNF, an important mediator of inflammatory and innate immune responses, can be regulated by binding to soluble TNF receptors. The 55-kDa type 1 TNFR (TNFR1), the key receptor for TNF signaling, is released to the extracellular space by two mechanisms, the inducible cleavage and shedding of 34-kDa soluble TNFR1 (sTNFR1) ectodomains and the constitutive release of full-length 55-kDa TNFR1 within exosome-like vesicles. The aim of this study was to identify and characterize TLR signaling pathways that mediate TNFR1 release to the extracellular space. To our knowledge, we demonstrate for the first time that polyinosinic-polycytidylic acid [poly (I:C)], a synthetic dsRNA analogue that signals via TLR3, induces sTNFR1 shedding from human airway epithelial (NCI-H292) cells, whereas ligands for other microbial pattern recognition receptors, including TLR4, TLR7, and nucleotide-binding oligomerization domain containing 2, do not. Furthermore, poly (I:C) selectively induces the cleavage of 34-kDa sTNFR1 ectodomains but does not enhance the release of full-length 55-kDa TNFR1 within exosome-like vesicles. RNA interference experiments demonstrated that poly (I:C)-induced sTNFR1 shedding is mediated via activation of TLR3-TRIF-RIP1 signaling, with subsequent activation of two downstream pathways. One pathway involves the dual oxidase 2-mediated generation of reactive oxygen species, and the other pathway is via the caspase-mediated activation of apoptosis. Thus, the ability of dsRNA to induce the cleavage and shedding of the 34-kDa sTNFR1 from human bronchial epithelial cells represents a novel mechanism by which innate immune responses to viral infections are modulated.     

IgG4 can induce an M2-like phenotype in human monocyte-derived macrophages through FcγRI

Antibodies evoke cellular responses through the binding of their Fc region to Fc receptors, most of which contain immunoreceptor tyrosine-based activation motif domains and are thus considered “activating.” However, there is a growing appreciation of these receptors for their ability to deliver an inhibitory signal as well. We previously described one such phenomenon whereby interferon (IFN)γ signaling is inhibited by immune complex signaling through FcγRI. To understand the implications of this in the context of therapeutic antibodies, we assessed individual IgG subclasses to determine their ability to deliver this anti-inflammatory signal in monocyte-derived macrophages. Like IgG1, we found that IgG4 is fully capable of inhibiting IFNγ-mediated events. In addition, F(ab’)2 fragments that interfere with FcγRI signaling reversed this effect. For mAbs developed with either an IgG1 or an IgG4 constant region for indications where inflammation is undesirable, further examination of a potential Fc-dependent contribution to their mechanism of action is warranted.     

Human monoclonal IgG rheumatoid factor has structural homology with bacterial Fc receptor proteins

A cloned lymphoblast cell line, hRF-1, that secreted human monoclonal IgG4 rheumatoid factor autoantibody was produced by Epstein-Barr virus transformation of lymphocytes from rheumatoid arthritis synovium. The binding of hRF-1 rheumatoid factor to IgG globulins of different mammalian species was similar to the binding specificity of Staphylococcus aureus protein A (SpA) and to antibodies found in the sera from patients with rheumatoid arthritis. hRF-1 also had the same binding pattern to human IgG subclasses as SpA. Direct competition was observed between SpA and hRF-1 in binding IgG Fc. These results provide evidence for structural homology between a bacterial Fc receptor protein (SpA) and the monoclonal IgG rheumatoid factor.     

IgG4 can induce an M2-like phenotype in human monocyte-derived macrophages through FcγRI

Antibodies evoke cellular responses through the binding of their Fc region to Fc receptors, most of which contain immunoreceptor tyrosine-based activation motif domains and are thus considered “activating.” However, there is a growing appreciation of these receptors for their ability to deliver an inhibitory signal as well. We previously described one such phenomenon whereby interferon (IFN)γ signaling is inhibited by immune complex signaling through FcγRI. To understand the implications of this in the context of therapeutic antibodies, we assessed individual IgG subclasses to determine their ability to deliver this anti-inflammatory signal in monocyte-derived macrophages. Like IgG1, we found that IgG4 is fully capable of inhibiting IFNγ-mediated events. In addition, F(ab’)2 fragments that interfere with FcγRI signaling reversed this effect. For mAbs developed with either an IgG1 or an IgG4 constant region for indications where inflammation is undesirable, further examination of a potential Fc-dependent contribution to their mechanism of action is warranted.     

Structural basis for Fc gammaRIIa recognition of human IgG and formation of inflammatory signaling complexes

The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. FcγRIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of FcγRIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of FcγRIIa (FcγRIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence FcγRIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for FcγRIIa (IV.3), FcγRIIb (X63-21), and a pan FcγRII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of FcγRIIa and FcγRIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of FcγRIIa-HR binds Ag-Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.     

Proteasome inhibition induces TNFR1 shedding from human airway epithelial (NCI-H292) cells

The type 1 55-kDa TNF receptor (TNFR1) is an important modulator of lung inflammation. Here, we hypothesized that the proteasome might regulate TNFR1 shedding from human airway epithelial cells. Treatment of NCI-H292 human airway epithelial cells for 2 h with the specific proteasome inhibitor clasto-lactacystin beta-lactone induced the shedding of proteolytically cleaved TNFR1 ectodomains. Clasto-lactacystin beta-lactone also induced soluble TNFR1 (sTNFR1) release from the A549 pulmonary epithelial cell line, as well as from primary cultures of human small airway epithelial cells and human umbilical vein endothelial cells. Furthermore, sTNFR1 release induced by clasto-lactacystin beta-lactone was not a consequence of apoptosis or the extracellular release of TNFR1 exosome-like vesicles. The clasto-lactacystin beta-lactone-induced increase in TNFR1 shedding was associated with reductions in cell surface receptors and intracytoplasmic TNFR1 stores that were primarily localized to vesicular structures. As expected, the broad-spectrum zinc metalloprotease inhibitor TNF-alpha protease inhibitor 2 (TAPI-2) attenuated clasto-lactacystin beta-lactone-mediated TNFR1 shedding, which is consistent with its ability to inhibit the zinc metalloprotease-catalyzed cleavage of TNFR1 ectodomains. TAPI-2 also reduced TNFR1 on the cell surface and attenuated the clasto-lactacystin beta-lactone-induced reduction of intracytoplasmic TNFR1 vesicles. This suggests that TNFR1 shedding induced by clasto-lactacystin beta-lactone involves the zinc metalloprotease-dependent trafficking of intracytoplasmic TNFR1 vesicles to the cell surface. Together, these data are consistent with the conclusion that proteasomal activity negatively regulates TNFR1 shedding from human airway epithelial cells, thus identifying previously unrecognized roles for the proteasome and zinc metalloproteases in modulating the generation of sTNFRs.     

Improved monovalent TNF receptor 1-selective inhibitor with novel heterodimerizing Fc

The development of alternative therapeutic strategies to tumor necrosis factor (TNF)-blocking antibodies for the treatment of inflammatory diseases has generated increasing interest. In particular, selective inhibition of TNF receptor 1 (TNFR1) promises a more precise intervention, tackling only the pro-inflammatory responses mediated by TNF while leaving regenerative and pro-survival signals transduced by TNFR2 untouched. We recently generated a monovalent anti-TNFR1 antibody fragment (Fab 13.7) as an efficient inhibitor of TNFR1. To improve the pharmacokinetic properties of Fab 13.7, the variable domains of the heavy and light chains were fused to the N-termini of newly generated heterodimerizing Fc chains. This novel Fc heterodimerization technology, designated “Fc-one/kappa” (Fc1κ) is based on interspersed constant Ig domains substituting the CH3 domains of a γ1 Fc. The interspersed immunoglobulin (Ig) domains originate from the per se heterodimerizing constant CH1 and CLκ domains and contain sequence stretches of an IgG1 CH3 domain, destined to enable interaction with the neonatal Fc receptor, and thus promote extended serum half-life. The resulting monovalent Fv-Fc1κ fusion protein (Atrosimab) retained strong binding to TNFR1 as determined by enzyme-linked immunosorbent assay and quartz crystal microbalance, and potently inhibited TNF-induced activation of TNFR1. Atrosimab lacks agonistic activity for TNFR1 on its own and in the presence of anti-human IgG antibodies and displays clearly improved pharmacokinetic properties.     

Aspirin-induced apoptosis in Jurkat cells is not mediated by peroxisome proliferator-activated receptor delta

Apoptosis-inducing agents have been reported to cause rapid shedding of tumor necrosis factor receptor 1 (TNFR1) in endothelial cells (EC). Oxidized LDL (oxLDL) has also been known to induce apoptosis of EC and to inhibit proliferation of EC. In the present study, we show that oxLDL also causes shedding of TNFR1 in EC and that EC transfected with soluble TNFR1 (sTNFR1 ), which is an extracellular domain of TNFR1, can antagonize the toxicity induced by oxLDL. These results suggest that transfection with the sTNFR1 gene plays a protective role against the injury of EC induced by oxLDL. We speculate therefore that sTNFR1 can be a new strategy for treatment of atherogenesis possibly by preventing shedding of TNFR1.     

Plasma membrane microdomains regulate TACE-dependent TNFR1 shedding in human endothelial cells

Upon stimulation by histamine, human vascular endothelial cells (EC) shed a soluble form of tumour necrosis factor receptor 1 (sTNFR1) that binds up free TNF, dampening the inflammatory response. Shedding occurs through proteolytic cleavage of plasma membrane-expressed TNFR1 catalysed by TNF-α converting enzyme (TACE). Surface expressed TNFR1 on EC is largely sequestered into specific plasma membrane microdomains, the lipid rafts/caveolae. The purpose of this study was to determine the role of these domains in TACE-mediated TNFR1 shedding in response to histamine. Human umbilical vein endothelial cells derived EA.hy926 cells respond to histamine via H1 receptors to shed TNFR1. Both depletion of cholesterol by methyl-β-cyclodextrin and small interfering RNA knockdown of the scaffolding protein caveolin-1 (cav-1), treatments that disrupt caveolae, reduce histamine-induced shedding of membrane-bound TNFR1. Moreover, immunoblotting of discontinuous sucrose gradient fractions show that TACE, such as TNFR1, is present within low-density membrane fractions, concentrated within caveolae, in unstimulated EA.hy926 endothelial cells and co-immunoprecipitates with cav-1. Silencing of cav-1 reduces the levels of both TACE and TNFR1 protein and displaces TACE, from low-density membrane fractions where TNFR1 remains. In summary, we show that endothelial lipid rafts/caveolae co-localize TACE to surface expressed TNFR1, promoting efficient shedding of sTNFR1 in response to histamine.     

Recurrent infections and immune evasion strategies of Staphylococcus aureus

Staphylococcus aureus causes purulent skin and soft tissue infections (SSTIs) that frequently reoccur. Staphylococal SSTIs can lead to invasive disease and sepsis, which are among the most significant causes of infectious disease mortality in both developed and developing countries. Human or animal infections with S. aureus do not elicit protective immunity against staphylococcal diseases. Here we review what is known about the immune evasive strategies of S. aureus that enable the pathogen's escape from protective immune responses. Three secreted products are discussed in detail, staphylococcal protein A (SpA), staphylococcal binder of immunoglobulin (Sbi) and adenosine synthase A (AdsA). By forming a complex with V(H)3-type IgM on the surface of B cells, SpA functions as a superantigen to modulate antibody responses to staphylococcal infection. SpA also captures pathogen-specific antibodies by binding their Fcγ portion. The latter activity of SpA is shared by Sbi, which also associates with complement factors 3d and factor H to promote the depletion of complement. AdsA synthesizes the immune signaling molecule adenosine, thereby dampening innate and adaptive immune responses during infection. We discuss strategies how the three secreted products of staphylococci may be exploited for the development of vaccines and therapeutics.     

Staphylococcus aureus protein A activates TACE through EGFR-dependent signaling

Among the many adhesins and toxins expressed by Staphylococcus aureus, protein A is an exceptionally complex virulence factor, known to interact with multiple eukaryotic targets, particularly those with immunological functions. Protein A acts as a ligand that can mimic TNF-alpha to activate TNFR1 and subsequent proinflammatory signaling. It also stimulates the cleavage of TNFR1 from the surface of epithelial cells and macrophages, which serves to limit TNF-alpha signaling. We characterized the signaling pathway responsible for TNFR1 shedding and identified protein A mutants which could activate TNFR1-dependent signaling, but were unable to activate TACE, the TNFR1 sheddase. Activation of TACE was dependent upon a discrete interaction between the previously defined IgG-binding domain of protein A and the epidermal growth factor receptor (EGFR), which in turn induced TACE phosphorylation through a c-Src-erk1/2-mediated cascade. This novel interaction was independent of the autocrine activation of EGFR and protein A-induced TGF-alpha was neither required nor sufficient to activate TNFR1 shedding. Thus, staphylococci exploit the ubiquitous and multifunctional EGFR to regulate the availability of TNFR1 on mucosal and immune cells.     

TNFR-Fc fusion protein expressed by in vivo electroporation improves survival rates and myocardial injury in coxsackievirus induced murine myocarditis

Tumor necrosis factor-alpha (TNF-alpha) is one of the major cytokines that modulate the immune response in viral myocarditis, but its role has not yet been thoroughly evaluated. We antagonized TNF-alpha using the expressed soluble p75 TNF receptor linked to the Fc portion of the human IgG1 gene (sTNFR:Fc) by in vivo electroporation, and evaluated its effects on experimental coxsackieviral B3 (CVB3) myocarditis. A plasmid DNA encoding sTNFR:Fc (15microg/mouse) was injected into the gastrocnemius muscles of Balb/C male mice followed by electroporation (day -1). Control mice were injected with an empty vector. One day after electroporation, mice were infected with CVB3 (day 0). Serum levels of sTNFR:Fc increased from day 2 and peaked at day 5 following electroporation. The heart virus titers of sTNFR:Fc mice were higher than those of controls at day 3. However, subsequent to day 12, the survival rates of the sTNFR:Fc mice were significantly higher than those of the controls (36% versus 0% at day 27, P<0.01). Histopathological examination indicated that inflammation and myocardial fibrosis were significantly decreased in sTNFR:Fc mice at day 12. The expressed sTNFR:Fc could modulate the inflammatory process during the post-viremic phase of viral myocarditis.     

Staphylococcal surface display of immunoglobulin A (IgA)- and IgE-specific in vitro-selected binding proteins (affibodies) based on Staphylococcus aureus protein A.

An expression system designed for cell surface display of hybrid proteins on Staphylococcus carnosus has been evaluated for the display of Staphylococcus aureus protein A (SpA) domains, normally binding to immunoglobulin G (IgG) Fc but here engineered by combinatorial protein chemistry to yield SpA domains, denoted affibodies, with new binding specificities. Such affibodies, with human IgA or IgE binding activity, have previously been selected from a phage library, based on an SpA domain. In this study, these affibodies have been genetically introduced in monomeric or dimeric forms into chimeric proteins expressed on the surface of S. carnosus by using translocation signals from a Staphylococcus hyicus lipase construct together with surface-anchoring regions of SpA. The recombinant surface proteins, containing the IgA- or IgE-specific affibodies, were demonstrated to be expressed as full-length proteins, localized and properly exposed at the cell surface of S. carnosus. Furthermore, these chimeric receptors were found to be functional, since recombinant S. carnosus cells were shown to have gained IgA and IgE binding capacity, respectively. In addition, a positive effect in terms of IgA and IgE reactivity was observed when dimeric versions of the affibodies were present. Potential applications for recombinant bacteria with redirected binding specificity in their surface proteins are discussed.     

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen-binding fragments (Fabs) of an mAb allow for high-avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half-life. To augment the effector functions or half-life of an IgG1 mAb, we constructed a novel “2Fc” mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral-like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6- to 130-fold, resulting in apparent affinity increases of 7- to 42-fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild-type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.     

Efficient expression and purification of human aglycosylated Fcγ receptors in Escherichia coli

Effector Fc gamma receptors (FcγRs) are expressed on the surface of a variety of cells of hematopoietic lineage and serve as a bridge between adaptive and innate immune responses. The interaction between immune complexes, formed by IgG class antibodies that are crosslinked with antigen, and FcγRs triggers signaling cascades that result in numerous cellular responses including the activation or donwregulation of cytotoxic responses, cytokine release, and antibody synthesis. Here, the extracellular domains of the human type I transmembrane FcγRs were expressed in Escherichia coli and their interactions to subclass IgGs (IgG1, IgG2, IgG3, and IgG4) antibodies were analyzed. Expression using fully synthetic E. coli codon optimized FcγR genes and optimization of sequences for N‐terminal translation initiation region through mRNA secondary structure prediction enabled us to achieve high yield of purified, bacterially expressed receptors, including FcγRI and FcγRIIIa which have not been successfully expressed in bacteria until now. The aglycosylated FcγRs showed similar IgG subclass binding selectivity compared to the respective glycosylated FcγRs expressed in mammalian cells. Biotechnol. Bioeng. 2010;107: 21–30. © 2010 Wiley Periodicals, Inc.     

Activating and inhibitory Fc gamma receptors in rheumatoid arthritis: from treatment to targeted therapies

Fc gamma receptors (Fc gammaRs) bind the constant Fc region of IgG molecules. IgG/antigen-containing immune complexes elicit a variety of effector functions in cells that express activating Fc gammaRs. Because activating Fc gammaRs are present on cells from the innate immune system, such as dendritic cells, monocytes/macrophages and granulocytes, these IgG receptors form a crucial link between the innate and the acquired immune systems. Recently, the ability to detect the inhibitory Fc gammaRIIb on cells has indicated an imbalance between activating and inhibitory Fc gammaRs in rheumatoid arthritis. This progress offers an opportunity to study modulation of Fc gammaR balance and could stimulate development of Fc gammaR-directed immunotherapy.     

Staphylococcus aureus protein A binding to von Willebrand factor A1 domain is mediated by conserved IgG binding regions

Protein A (Spa) is a surface-associated protein of Staphylococcus aureus best known for its ability to bind to the Fc region of IgG. Spa also binds strongly to the Fab region of the immunoglobulins bearing V(H)3 heavy chains and to von Willebrand factor (vWF). Previous studies have suggested that the protein A-vWF interaction is important in S. aureus adherence to platelets under conditions of shear stress. We demonstrate that Spa expression is sufficient for adherence of bacteria to immobilized vWF under low fluid shear. The full length recombinant Ig-binding region of protein A, Spa-EDABC, fused to glutathione-S-transferase (GST), bound recombinant vWF in a dose-dependent and saturable fashion with half maximal binding of about 30 nm in immunosorbent assays. Full length-Spa did not bind recombinant vWF A3 domain but displayed binding to recombinant vWF domains A1 and D'-D3 (half maximal binding at 100 nm and 250 nm, respectively). Each recombinant protein A Ig-binding domain bound to the A1 domain in a similar manner to the full length-Spa molecule (half maximal binding 100 nm). Amino acid substitutions were introduced in the GST-SpaD protein at sites known to be involved in IgG Fc or in V(H)3 Fab binding. Mutants altered in residues that recognized IgG Fc but not those that recognized V(H)3 Fab had reduced binding to vWF A1 and D'-D3. This indicated that both vWF regions recognized a region on helices I and II that overlapped the IgG Fc binding site.     

IgG binding kinetics to oligo B protein A domains on lipid layers immobilized on a 27 MHz quartz-crystal microbalance

Although molecular recognitions between membrane receptors and their soluble ligands have been analyzed using their soluble proteins in bulk solutions, molecular recognitions of membrane receptors should be studied on lipid membranes considering their orientation and dynamics on membrane surfaces. We employed Staphylococcal Protein A (SpA) oligo B domains with long trialkyl-tags from E. coli (LppBx, x = 1, 2, and 5) and immobilized LppBx on lipid layers using hydrophobic interactions from the trialkyl-tag, while maintaining the orientation of B domain-chains on a 27 MHz quartz-crystal microbalance (QCM; AT-cut shear mode). The binding of IgG Fc regions to LppBx on lipid layers was detected by frequency decreases (mass increases) on the QCM. The maximum amount bound (Delta m(max)), association constants (K(a)), association and dissociation rate constants (k(1) and k(-1), respectively) were obtained. Binding kinetics of IgG to LppB2 and LppB5 were quite similar, showing a simple 1:1 binding of the IgG Fc region to the B domain, when the surface coverage of LppB2 and LppB5 on the lipid surface is low (1.4%). When LppB5 was immobilized at the high surface coverage of 3.5%, the complex bindings of IgG such as one IgG bound to one or two LppB5 on the membrane could be observed. IgG-LppB1 binding was largely restricted because of steric hindrance on lipid surfaces. This gives a suggestion why Protein A has five IgG binding domains.     

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.