Receptor CD32A Γ Low-Affinity Fc Expressing R and H Allelic Forms Of

The Journal of Immunology

Dynamics of the Interaction of Human IgG Subtype Immune Complexes with Cells Expressing R and H Allelic Forms of a Low-Afﬁnity Fc␥ Receptor CD32A1

Rangaiah Shashidharamurthy,* Fang Zhang,† Aaron Amano,* Aparna Kamat,* Ravichandran Panchanathan,* Daniel Ezekwudo,* Cheng Zhu,† and Periasamy Selvaraj2*

CD32A, the major phagocytic Fc␥R in humans, exhibits a polymorphism in the ligand binding domain. Individuals homozygous for the R allelic form of CD32A (CD32AR allele) are more susceptible to bacterial infections and autoimmune diseases as compared with H allelic CD32A (CD32AH) homozygous and CD32AR/H heterozygous individuals. To understand the mechanisms behind this differential susceptibility, we have investigated the dynamics of the interaction of these allelic forms of CD32A when they are simultaneously exposed to immune complexes (IC). Binding studies using Ig fusion proteins of CD32A alleles showed that the R allele has significantly lower binding not only to human IgG2, but also to IgG1 and IgG3 subtypes. Competition assays using purified molecules demonstrated that CD32AH-Ig outcompetes CD32AR-Ig for IC binding when both alleles simultaneously compete for the same ligand. CD32AH-Ig blocked the IC binding mediated by both the allelic forms of cell surface CD32A, whereas CD32AR-Ig blocked only CD32AR and was unable to cross-block IC binding mediated by CD32AH. Two-dimensional affinity measurements also demonstrated that CD32AR has significantly lower affinity toward all three subtypes as compared with CD32AH. Our data suggest that the lower binding of CD32AR not only to IgG2 but also to IgG1 and IgG3 might be responsible for the lack of clearance of IC leading to increased susceptibility to bacterial infections and autoimmune diseases. Our data further suggests that in humans, inflammatory cells from CD32AR/H heterozygous individuals may predominantly use the H allele to mediate Ab-coated target cell binding during phagocytosis and Ab-dependent cellular cytotoxicity, resulting in a phenotype similar to CD32AH homozygous individuals. The Journal of Immunology, 2009, 183: 8216–8224.

mong the receptors for the Fc domain of immune com- bacteria in humans including human pathogens such as Neisseria plexes (IC),3 the low-affinity Fc␥R play a central role meningitidis, Haemophilus influenzae, and Streptococcus pneu- A in many types of Ab-dependent cellular cytotoxicity moniae (16–18). Yee et al. (19) showed that 90% of CD32AR and immunophagocytosis (1–5). In humans, CD32A, which is a homozygous individuals are more susceptible to pneumococcal Fc␥RII, is the major phagocytic Fc receptor (6). Human CD32A infection. has low affinity for monomeric IgG, but it binds stably to IC. Apart from bacterial infections, CD32AR allele is also associ- CD32A has been shown to exhibit a polymorphism in the ligand ated with susceptibility to the development of certain autoimmune binding domain. This single nucleotide polymorphism in the li- disease such as systemic lupus erythematosus (SLE) (16, 20–24). gand binding domain causes a substitution of amino acid arginine Various clinical studies have shown that patients with SLE who R H (CD32A ) to histidine (CD32A ) at position 131. Both CD32A are CD32AR have a higher likelihood of developing proteinurea, R alleles bind to human IgG1 and IgG3, but the CD32A allotype hemolytic anemia, antinuclear RNP Abs, glomerulonephritis and shows a lower binding for human IgG2 when compared with hypocomplementenia (25). Development of SLE at a younger age H R CD32A (7–9). Evidence suggests that CD32A allele is associ- was reported in patients with the CD32AR genotype, with an ear- ated with increased susceptibility to bacterial infections (10–15). lier incidence of arthritis, sicca syndrome, nephritis, lymphadeni- Human IgG2 is the major subclass of Ab elicited by encapsulated tis, hemotologic abnormalities, lupus anticoagulant, cryoglobu- linemia, and hypocomplementemia (25). *Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA Taken together, these studies suggest that the CD32A polymor- 30322; and †Department of Biomedical engineering, Georgia Institute of Technology, phism plays a pivotal role in certain infectious and autoimmune Atlanta, GA 30332 diseases. The increased susceptibility of CD32AR homozygous in- Received for publication August 4, 2009. Accepted for publication October 10, 2009. dividuals for the observed diseases may be due to the poor clear- The costs of publication of this article were defrayed in part by the payment of page ance of IC. Homozygous CD32AH individuals, in contrast, are not charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. susceptible to certain bacterial infections and autoimmune diseases 1 This study was supported by Grants R01 AI049400 and R21HL09126S from the because IC is cleared efficiently (20, 21, 26, 27). Interestingly, National Institutes of Health (to P.S.), a grant from Emory University Seed Grant CD32AR/H heterozygous individuals are also resistant to certain Program (to P.S.), and by Grant R01 AI38282 from the National Institutes of Health R (to C.Z.). bacterial infections even though they express the CD32A allele. R 2 Address correspondence and reprint requests to Dr. Periasamy Selvaraj, Department It is not clear why the coexpression of CD32A in heterozygous H of Pathology, Emory University School of Medicine, 7309 Woodruff Memorial Build- individuals is not reducing the efficiency of CD32A allele. We ing, 101 Woodruff Circle, Atlanta, GA 30322. E-mail address: [email protected] hypothesize that in heterozygous individuals, CD32AH outcom- 3 Abbreviations used in this paper: IC, immune complex; CHO, Chinese hamster petes CD32AR for ligand binding when both alleles are expressed ovary; SLE, systemic lupus erythematosus. on the same cell. To test our hypothesis, we have analyzed the Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 interaction of IC with cells expressing R and H allelic forms of www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902550 The Journal of Immunology 8217

CD32A and their competition for ligand binding using recombi- were washed and HRP substrate was added. The reaction was stopped by nant dimeric forms of soluble R and H forms of CD32A alleles. adding1NH2SO4 and read at 450 nm. The HRP-IC of human IgG sub- The results presented in this study demonstrate that CD32AH out- types bound to IV.3, a blocking mAb for human CD32A molecule, treated R CD32A-Ig coated wells were taken as nonspecific binding. competes CD32A when they simultaneously compete for the For FITC-labeled IC binding assay, the Fc␥R-positive cells (50 ␮lof H same ligand. Such a dominance of CD32A allele in heterozygous 5 ϫ 106/ml) were incubated with FITC-labeled IC of human IgG subtypes individuals may be due to the higher affinity of CD32AH for all (10 ␮g/ml) in binding buffer for1hat4°Cintheabsence or presence of human IgG isotypes as compared with CD32AR, which is demon- purified CD32A-Ig molecules or blocking mAbs. Cells treated with IV.3 mAb for 30 min at 4°C, served as a specificity control. Cells were then strated in this study by cell binding assays and two-dimensional washed and resuspended in 150 ␮l of binding buffer and fixed by adding affinity measurement studies. 150 ␮l of 2% formalin in PBS. Binding of FITC-labeled IC to Fc␥R- positive cells was analyzed by flow cytometry. Materials and Methods Particulate IC binding assay Cell lines and reagents SRBC were coupled with human IgG subtypes (referred to in the work as PKH-26 labeling kit, rabbit anti-DNP IgG, HRP-conjugated anti-human Fc EA) by the chromium chloride method (34). PKH labeling of EA was Ј Ab, HRP- and FITC-conjugated goat anti-human IgG F(ab )2 specific for L performed using a PKH labeling kit. Binding of Fc␥R-positive cells to chain of human IgG, and cyanogen bromide-activated Sepharose were PKH-labeled EA was analyzed by flow cytometry (28, 32). Briefly, PKH- from Sigma-Aldrich. Human IgG subtypes were from Sigma-Aldrich. As labeled EA were washed and resuspended in binding buffer. If aggregates per the manufacturer’s data sheet, the purity of the IgG molecules is of EA were noticed, they were removed using 75-␮m nylon filters before Ͼ95%. To avoid the problems associated with the storage of IgG mole- the assay. Fc␥R-positive cells (50 ␮lof5ϫ 106/ml) in binding buffer were cules in solution, the molecules were immediately aliquoted and stored at incubated with PKH-labeled EA (50 ␮lof1.5ϫ 108/ml) for2hat4°C. Ϫ20°C. The ICs were made fresh and used for the experiments. We have Binding assays were performed in the absence or continuous presence of also used IgG molecules from two different lots that were found to have CD32A-Ig molecules or 10 ␮g/ml mAbs for CD32A (IV.3) during the similar binding to human Fc␥R. The following IgG molecules (with cat- incubation with EA. EA bound to cells were analyzed by flow cytometry. alog number) were purified from human plasma by combination of chro- PKH-labeled unopsonized erythrocytes were used as a control in all the matographic techniques as per Sigma-Aldrich: IgG1 (I5154), IgG2 (I5404), experiments. Mean fluorescent intensity at FL2 channel and the percentage IgG3 (I5654), and IgG4 (4639). FITC- and Cy5-conjugated goat anti- of Fc␥R-positive cells bound to EA were determined using FACScan flow Ј human Fc-specific IgG F(ab )2 from Jackson ImmunoResearch Laborato- cytometer (BD Biosciences). The binding index was calculated using the ries. Lipofectamine 2000 was from Invitrogen. The Micro BCA-protein formula for the percentage of cells bound to EA ϫ mean fluorescence assay kit was purchased from Pierce and the HRP substrate from Bio-Rad. divided by 100. QuikChange II Site-Directed Mutagenesis kit was from Stratagene. Chi- nese hamster ovary (CHO) cells (clone K1) were from American Type Competition assay Culture Collection. Human RBC were isolated from healthy volunteers. Saturating concentration of CD32A-Ig molecules for rabbit anti-DNP IgG SRBC were from Colorado Serum Company. Cell culture reagents were opsonized sheep erythrocytes (EA) binding was determined using flow from Life Technologies. cytometry. Briefly, sheep erythrocytes were coated with DNP as described Construction, expression, and purification of recombinant (35) and opsonized with rabbit anti-DNP IgG (EA). EA (50 ␮lof1ϫ 108 soluble CD32A-Ig molecules cells/ml) were incubated with various concentrations of CD32A-Ig molecules for1hat4°C. After washing twice with binding buffer the FITC- R Ј The construction and expression of the dimeric form of CD32A -Ig was conjugated F(ab )2 goat anti-human Fc-specific IgG (Ig portion of CD32A-Ig conducted by ligating the extracellular domain of human CD32AR to molecules is a human IgG1 Fc domain) was added and incubated for an- the Fc domain of the human IgG1 H chain as described earlier (28). The other1hat4°C. The cells were washed twice and analyzed for CD32A-Ig mutated IgG1 Fc CH2-CH3 domains were obtained from Dr. P. Linsley molecules binding. For the competition assay, EA (50 ␮lof1ϫ 108 cells/ (Bristol-Meyers Squibb, Seattle, WA). Mutations in the Fc domain are ml) were incubated with 50 ␮l of CD32A-Ig molecules either alone or in L267F, L268E, G270A, and A363T (numbered as in accession number combination (equimolar or saturating concentration) for1hat4°C. The Ј AAH69020.1). These mutations have been shown to abolish the binding CD32A-Ig molecules were preincubated with F(ab )2 goat anti-human Fc- of Fc␥R (29–31). The CD32AH-Ig was constructed by QuikChange II specific Ab conjugated with Cy5 (for CD32AH-Ig) or FITC (for CD32AR- Site-Directed Mutagenesis kit (Stratagene) using CD32AR-Ig DNA as a Ig)for1hat4°C. The secondary Ab was titrated and excess dimers were template. The recombinant molecules were purified from CHO cell added to avoid the cross-binding of secondary Ab during the incubation of transfectants using a protein G-Sepharose column (Pharmacia Biotech) both the dimers with EA. The cells were washed and analyzed for (32). The immunoaffinity purified CD32A-Ig molecules were analyzed CD32A-Ig molecules binding. The cells treated with the secondary Ab using 10% SDS-PAGE under reducing and nonreducing conditions and alone served as a specificity control. the protein bands were visualized by silver staining and Western blot Micropipette adhesion frequency assay analysis. Protein concentration was measured by Micro BCA protein assay kit (Pierce) using BSA as a standard. To determine two-dimensional affinity (the affinity of a receptor-ligand pair expressed on opposing cell surface) of CD32A alleles for the human IgG Establishment of CHO stable transfectants expressing cell subtypes, the micropipette adhesion frequency assay was conducted as de- surface CD32A alleles scribed (34). Briefly, human RBC were coated with human IgG subtypes using CrCl coupling method as described. The site density of human IgG CHO cells were transfected with pUB6A plasmid vectors containing 3 subtypes on human RBCs and Fc receptors on CHO cells was determined CD32AR and CD32AH gene using Lipofectamine. Forty-eight hours after by flow cytometry using fluorescence-coupled standard beads (Quantum 25 transfection, cell culture medium containing 20 ␮g/ml blasticidin (Life FITC High Level; Bangs Laboratory). Technologies) was used as the selection medium to establish the stable The micropipette apparatus used in this experiment has been previ- transfectants of CHO-CD32A. Cells expressing high levels of transfected ously described (34, 36, 37). Briefly, a micropipette-aspirated RBC molecules were selected by a panning procedure (33). coated with human IgG subtypes was driven by a piezoelectric trans- Soluble IC binding assay lator to make a 10 s contact with a CHO cell expressing CD32A alleles held stationary by another pipette. At the end of the contact duration, Soluble IC was prepared by mixing with either HRP- or FITC-conjugated the two cells were separated by retracting the RBC. Upon retraction, an Ј ␬ F(ab )2 goat anti-human IgG specific for L chain ( ) with human IgG adhesion event between the two cells was indicated by stretched RBC subtypes (1:1 molar ratio) for4hat4°C. HRP- and FITC-conjugated membrane (see Fig. 6A). This contact test cycle was repeated 100 times Ј F(ab )2 goat anti-human IgG were used for ELISA and cell binding assays, using the same pair of cells, keeping the contact duration (t) and the ap- ϳ ␮ Ϫ2 ء respectively. The complex was centrifuged at 15,000 rpm for 30 min at 4°C parent area (Ac 3 m ) constant, and the number of adhesion event and the supernatant was used for the soluble IC binding assay. For ELISA, was counted to obtain an adhesion frequency (Pa). Nonspecific binding is CD32A-Ig molecules (50 ␮lof10␮g/ml) were coated on plates overnight determined using RBC coated with BSA. The effective binding affinity ϭ Ϫ and wells were blocked for 1 h with binding buffer (PBS/5 mM EDTA with (AcKa) and off-rate (kr) were extracted by fitting the equation, Pa 1 ␮ Ϫ Ϫ Ϫ 1% BSA (pH 7.4)). The HRP-IC of human IgG subtypes (50 lof10 exp( mrmlAcKa[1 exp( krt)]), in which mr and ml are receptor and ␮g/ml) was then added, and incubation continued for1hat4°C. The wells ligand site density, respectively. For experiments in this study, the adhesion 8218 INTERACTION OF CD32A ALLELES WITH IMMUNE COMPLEXES

FIGURE 1. Detection of CD32A-Ig dimers. SDS-PAGE analysis of immunoaffinity purified dimeric CD32AR-Ig and CD32AH-Ig alleles. Dimers were purified using gamma bind plus column from CHO cell culture supernatant. The purified protein (5 ␮g) was subjected to SDS-PAGE under reducing (left) and nonreducing (right) conditions, and the protein bands were visualized using the silver staining method (A) and Western blotting (B).

frequency was determined at a sufficiently long contact duration (t ϭ 10 s: bound to human IgG1, IgG2 and IgG3, whereas CD32AR-Ig bound Ϫ Ϸ exp( krt) 0) without measuring a full binding curve to simply estimate to IgG1 and IgG3 but not to IgG2 (Fig. 2). Both alleles were ϭ the effective binding affinity (AcKa) from the equation, AcKa 1/mrml Ϫ Ϫ1 3 ϱ unable to bind to IgG4. The wells with CD32A-Ig molecules and (ln(1 Pa) ), which is derived from the steady state version (i.e., t ) of the adhesion frequency (P ) equation. treated with IV.3 mAb (a mAb that specifically blocks binding of a Ј IC to CD32A) before the addition of HRP-F(ab )2-labeled IC were Ј Statistical analysis used as a specificity control. The results show that HRP-F(ab )2- A statistical comparison of the CD32AR and CD32AH alleles was per- labeled IC binding to CD32A is specific and can be completely formed using the Student t test. Values for p Ͻ 0.01 were considered as blocked by IV.3 mAb. This also rules out the possibility of un- Ͻ Ј significant and p 0.001 considered highly significant. complexed HRP-F(ab )2 binding to CD32A-Ig molecules. Next, we determined whether the dimerized CD32A alleles Results had a ligand binding pattern similar to cell surface CD32A alleles. To Recombinant Ig fusion proteins of CD32A alleles are secreted test this possibility, we assayed the CHO transfectants expressing the as a homodimers CD32AR and CD32AH alleles separately. Flow cytometry analysis using IV.3 mAb confirmed that both alleles of CD32A were ex- The extracellular domain of CD32AR was ligated to the mu- pressed to almost similar levels on CHO cell surface (Fig. 3A). tated CH2 and CH3 regions of the Fc domain of human IgG1 Soluble IC of human IgG subtypes conjugated with FITC was molecule using a strategy similar to the construction of prepared as described in Materials and Methods. Flow cytometry CD16A-Ig (28). The CD32AH-Ig was created by site-directed mutagenesis using CD32AR-Ig DNA as a template. The result- ant chimeric cDNA was transfected in CHO cells. The culture supernatant obtained from CHO transfectants was analyzed by a sandwich ELISA using mAb IV.3 (anti-CD32A mAb) to con- firm expression of CD32A (data not shown). Clones secreting high levels of CD32A-Ig fusion proteins were chosen for further characterization. CD32A-Ig molecules from cell culture supernatant were purified by a single step immunoaffinity chro- matography using protein G-Sepharose. The yield was nearly 1.5 mg for CD32AR-Ig and 2.0 mg for CD32AH-Ig per liter of culture supernatant. SDS-PAGE (Fig. 1A) and Western blot (Fig. 1B) analysis of the purified CD32A-Ig molecules showed a major band of 141 kDa and a minor band of ϳ120 kDa under nonreducing conditions and 68 kDa under reducing conditions, FIGURE 2. Binding of CD32A-Ig molecules to soluble IC (sIC). A 96- suggesting that the recombinant CD32A-Ig molecules are se- well plate was coated with 50 ␮l (10 ␮g/ml) CD32A-Ig molecules over- creted as a disulfide-linked homodimer. The minor protein band night at 4°C and wells were blocked with binding buffer (PBS/5 mM of 120 kDa (under nonreducing conditions) may be the precur- EDTA with 1% BSA). After washing the wells three times with binding sor or degradation product of CD32A-Ig because it reacted with buffer, 50 ␮l of HRP-conjugated soluble IC of human IgG subtypes (5 anti-human Fc-specific IgG in Western blot analysis (Fig. 1B). ␮g/ml) were added and incubated for another1hat4°C. Wells were washed three times; color was developed by adding the HRP substrate The IgG binding properties of CD32A-Ig molecules are similar and read at 450 nm. IV.3-treated wells served as a specificity control. to cell surface expressed CD32A alleles The ELISA OD readings after blocking with IV.3 mAb were as follows: CD32AH-Ig coated wells: IgG1 ϭ 0.017, IgG2 ϭ 0.019, IgG3 ϭ 0.016, The purified dimeric CD32A-Ig molecules were assayed for their IgG4 ϭ 0.018, and for CD32AR-Ig coated wells: IgG1 ϭ 0.011, IgG2 ϭ functional ability to bind soluble IC of human IgG subtypes. Sol- 0.015, IgG3 ϭ 0.017, IgG4 ϭ 0.02. The values are average of triplicate uble IC of human IgG subtypes conjugated with HRP were pre- readings and representative of three individual experiments. The values p Ͻ 0.01 and ,ء .pared as described in Materials and Methods and assayed for bind- are comparable with BSA coated negative control wells .p Ͻ 0.001 ,ءء ing to Fc␥R-Ig coated on the plates. As expected, CD32AH-Ig The Journal of Immunology 8219

FIGURE 3. Flow cytometry analysis of expression of CD32A alleles in CHO cells. Stable transfectants of CHO cells expressing CD32A alleles were stained with anti-hCD32A mAb (IV.3). A, Isotype control (open histogram) and the binding of IV.3 Ab to CD32A alleles (filled histogram) are indicated. Dotted line indicates the expression level of CD32A alleles. B, Binding of cell surface CD32A alleles to soluble IC (sIC) of human IgG subtypes. CHO cells expressing CD32A alleles were incubated with FITC-conjugated soluble IC of human IgG subtypes (10 ␮g/ml) in the presence and absence of IV.3 (10 ␮g/ml) and analyzed for FITC-labeled IC binding to CHO-CD32A alleles using flow cytometry (filled histogram). IV.3-treated cells served as a negative control (open histogram). Data are representative of three individual experiments. C, Representation of human IgG subtype soluble IC p Ͻ 0.001. D, Binding of cell surface ,ءء ,p Ͻ 0.01 ,ء .bound to CHO-CD32A alleles is shown. Results are mean Ϯ SD from three experiments CD32A alleles to human IgG subtypes coated with sheep erythrocytes (EA). CHO cells expressing CD32A alleles were incubated with human IgG subtype coated, PKH-labeled EA (50 ␮lof1.5ϫ 108 cells/ml) for2hat4°C. Results are mean Ϯ SD from three experiments. EA bound to the cells was analyzed by flow cytometry. Cells incubated with PKH-labeled unopsonized-E and IV.3-treated cells served as specificity control. The binding index was calculated using the formula for the percentage of cells bound to EA ϫ mean fluorescence divided by 100. Results are mean Ϯ .p Ͻ 0.001 ,ءء p Ͻ 0.01 and ,ء .SD of data from three experiments analysis of FITC-conjugated IC showed that CHO cells expressing tern of CD32A alleles to particulate IC of human IgG subtypes CD32AH allele (CHO-CD32AH) bound to human IgG1, IgG2, and using CHO cells expressing CD32AR and CD32AH forms. As IgG3, whereas CHO-CD32AR bound to IgG1 and IgG3 but not to shown in Fig. 3D, CHO cells expressing CD32AH alleles bound IgG2 (Fig. 3B, filled histogram). As a specificity control for FITC- to human IgG1, IgG2, and IgG3, whereas CHO-CD32AR cells Ј conjugated F(ab )2 binding, the CHO-CD32A cells were treated bound to IgG1 and IgG3 but not to IgG2. Neither of the alleles with IV.3 Ab before the addition of FITC-labeled IC. Results bound to IgG4. These results show that CD32AR and CD32AH show that the FITC-labeled IC was completely blocked by IV.3, alleles have a similar binding pattern for both soluble and par- Ј suggesting that the uncomplexed FITC-conjugated F(ab )2 does ticulate IC of human IgG subtypes. Soluble and particulate IC not bind nonspecifically to CD32A expressed on CHO cells binding to CD32A alleles was completely blocked by IV.3 mAb (Fig. 3B, open histogram). Fig. 3C represents the level of human (a mAb to CD32A) and served as a specificity control. These IgG subtypes IC bound to CHO-CD32A alleles. Thus, as we ob- results suggest that dimeric recombinant CD32A-Ig molecules served in the previous experiment with dimerized CD32A-Ig (Fig. and cell surface CD32A alleles interact similarly with human 2), both of the cell surface CD32A alleles bind to IgG1 and IgG3, IgG subtypes. Furthermore, dimerization of the recombinant but CD32AH binds better than CD32AR (Fig. 3C). CD32A-Ig molecules did not change its binding specificity to The ICs formed between autoantibodies and target cells are human IgG subtypes. Interestingly though, both CD32A alleles particulate in nature. Therefore, we determined the binding pat- bound to IgG1 and IgG3, CD32AH allele binding was better 8220 INTERACTION OF CD32A ALLELES WITH IMMUNE COMPLEXES

FIGURE 4. Competition of CD32AR-Ig and CD32AH-Ig for binding to rabbit IgG opsonized SRBCs. A, To determine the saturating concentration of CD32A-Ig molecules binding to rabbit IgG-coated erythrocytes (EA), varying concentrations of CD32A-Ig molecules were incubated separately with 50 ␮ ϫ 8 Ј lofEA(2 10 cells/ml) for 1h at 4°C. After washing three times with binding buffer, the incubation was continued with F(ab )2 goat anti-human Fc-specific Ab conjugated with FITC for1hat4°C. Cells were analyzed for CD32A-Ig molecules binding by flow cytometry. B, For competition assay, an equal amount or saturating concentration of both the molecules was mixed and incubated with EA. The CD32A-Ig molecules were preincubated with Ј H R F(ab )2 goat anti-human Fc-specific Ab conjugated with Cy5 (for CD32A -Ig) or FITC (for CD32A -Ig) for1hat4°Candthen added to EA to determine the competitive binding of both the alleles to EA. The cells were washed and analyzed for CD32A-Ig binding by flow cytometry. The cells treated with secondary Ab alone served as a specificity control. EA treated with secondary Ab alone (panel I), EA incubated with 50 ␮g/ml FITC-CD32AR-Ig (panel II), EA incubated with 50 ␮g/ml Cy5-conjugated CD32AH-Ig (panel III), EA incubated with CD32AR-Ig (50 ␮g/ml) and CD32AH-Ig (25 ␮g/ml) (panel IV), EA incubated with CD32AR-Ig (25 ␮g/ml) and CD32AH-Ig (25 ␮g/ml) (panel V), and EA incubated with CD32AR-Ig (50 ␮g/ml) and CD32AH-Ig (50 ␮g/ml) (panel VI). Unstained cells (lower left quadrant), CD32AR-Ig bound EA (lower right quadrant), CD32AH-Ig bound EA (upper left quadrant), and EA bound to both the CD32A-Ig molecules (upper right quadrant) are shown. Data are representative of three individual experiments. than the CD32AR allele. Taken together, these results suggest CD32A-Ig molecules) or saturating concentrations (CD32AR-Ig: that CD32A alleles not only differ in binding to IgG2 but also 50 ␮g/ml, CD32AH-Ig: 25 ␮g/ml) of CD32A-Ig molecules. to IgG1 and IgG3. CD32AH-Ig was pre-complexed with Cy5-conjugated goat anti- Ј R human Fc-specific F(ab )2 Ab, whereas CD32A -Ig was pre-com- CD32AH allele outcompetes CD32AR allele for ligand binding Ј plexed with FITC-conjugated goat anti-human Fc-specific F(ab )2 when both alleles are exposed to IC simultaneously Ab. Both molecules (either equimolar or saturating concentration) Because previous ligand binding experiments suggest that were then added to the EA to study the competition of these mol- CD32AH binds to IC more efficiently than CD32AR allele, next ecules for ligand binding. The secondary Ab was titrated, and ex- we studied whether CD32AH allele competes with CD32AR al- cess dimers were added to avoid cross-binding of the secondary lele for ligand binding when both the alleles are exposed to IC Ab to the dimers during the incubation of the dimers with EA. simultaneously. We studied the competition between these al- Both CD32AR-Ig molecules (Fig. 4B, panel II) and CD32AH-Ig leles for ligand binding using purified CD32A-Ig molecules. A molecules (Fig. 4B, panel III) bind to rabbit IgG-coated SRBC simple in vitro competition assay was performed to directly when incubated separately. When both molecules were incubated to- determine whether CD32AH competes with CD32AR for ligand gether at their saturation concentrations (CD32AR-Ig: 50 ␮g/ml, binding. In this experiment, we used rabbit IgG coated SRBC CD32AH-Ig: 25 ␮g/ml) (Fig. 4B, panel IV), 60% of the EA were (EA) as a model IC and anti-human Fc specific Ab for detec- Cy5-positive, indicating that 60% of EA bound only to CD32AH-Ig. tion. First, we determined the saturating concentration of the Only 40% of the EA were both Cy5- and FITC-positive, suggesting dimers binding to rabbit IgG coated SRBC by incubating rabbit that 40% of EA bound to both the dimers. At equimolar concentra- IgG coated SRBC with various concentration of CD32AR-Ig tions (25 ␮g/ml of both molecules) (Fig. 4B, panel V) (50 ␮g/ml of H Ј and CD32A -Ig separately. FITC-conjugated F(ab )2 goat anti- both molecules) (Fig. 4B, panel VI) of the dimers, more than 80% of human Fc-specific Ab was used to detect binding via flow cy- EA were Cy5-positive, indicating that more than 80% of EA was tometry. As shown in Fig. 4A, CD32AH-Ig reaches saturation bound only to CD32AH-Ig. Only less than 20% of the EA were both around 25 ␮g/ml, whereas CD32AR-Ig requires 50 ␮g/ml. These Cy5- and FITC-positive, suggesting that less than 20% of EA bound results suggest that these molecules differ significantly ( p Ͻ to both the dimers. The EA treated with secondary Ab alone served as 0.001) in their affinity for rabbit IgG binding, which is consistent a specificity control (Fig. 4B, panel I). These results suggest that with our previous report (38). Then we determined the competition CD32AH allele has a higher binding efficiency for IC and thus out- between these molecules for binding to rabbit IgG when both the competes the R allelic form for ligand binding under situations in alleles are exposed simultaneously to IC. In this experiment, we which both alleles are exposed to the same ligand simultaneously, as incubated the EA with either equimolar (25 ␮g/ml of each may happen in R/H heterozygous individuals. The Journal of Immunology 8221

FIGURE 5. Soluble CD32A-Ig molecules compete with CD32A alleles expressed on CHO cell surface and block binding of soluble IC (sIC) of human IgG subtypes. A, CHO-CD32AR (top) or CHO-CD32AH (bottom) cells were incubated with FITC-conjugated IC (soluble IC; 10 ␮g/ml) in the presence and absence of CD32A-Ig molecules (50 ␮g/ml) and analyzed. IV.3 Ab-treated cells served as a specificity control. Blocking reagents are represented on p Ͻ 0.001. B, Soluble CD32A-Ig molecules compete with cell surface ,ءء p Ͻ 0.01 and ,ء .the y-axis. Results are mean Ϯ SD from three experiments CHO-CD32A alleles and block binding of erythrocytes coated with human IgG subtypes. CHO-CD32AR (top) or CHO-CD32AH (bottom) cells were incubated human IgG subtype coated, PKH-labeled EA (50 ␮lof1.5ϫ 108 cells/ml) for2hat4°Cinthepresence and absence of purified CD32A-Ig molecules (50 ␮g/ml). The cells were analyzed for binding of EA using flow cytometry. IV.3-treated cells and cells incubated with PKH-labeled unopsonized SRBC served as a specificity control. Blocking reagents are represented on the y-axis. The binding index was calculated using the formula for the .p Ͻ 0.001 ,ءء p Ͻ 0.01 and ,ء .percentage of cells bound to EA ϫ mean fluorescence divided by 100. Results are mean Ϯ SD from three experiments

CD32AH-Ig is able to cross-block IC binding mediated by cell CD32AH allele has higher affinity for all human IgG subtypes surface CD32AR than CD32AR allele H Next we determined whether the CD32A -Ig molecules can The above ligand binding studies and cross-blocking studies sug- cross-block the ligand binding mediated by the cell surface gest that the CD32A alleles might differ in their affinity not only R CD32A alleles. The cross-blocking studies were conducted us- for human IgG2 but also for IgG1 and IgG3. Using a micropipette ing CHO transfectants expressing CD32AR H and CD32A alleles adhesion frequency assay, we measured the two-dimensional binding separately. In this experiment, we have used human IgG1 and affinity of the two CD32A alleles (CHO-CD32AR and CHO- IgG3 for CHO-CD32AR, whereas IgG1, IgG2, and IgG3 were CD32AH) for four human IgG subtypes (IgG1, IgG2, IgG3, and used for CHO-CD32AH for both soluble and particulate IC. As ␮ H IgG4). The adhesion frequency of CHO cells expressing either shown in Fig. 5A,at25 g/ml, CD32A -Ig was able to block R H more than 90% of human IgG subtype binding to both the cell CD32A or CD32A to RBC coated with one of the four subtypes of surface CD32A alleles. At the same concentration, CD32AR-Ig human IgG were measured and the effective binding affinity was cal- was able to block up to 90% of human IgG binding to CHO- culated as described in Materials and Methods. The typical binding is H CD32AR, but only cross-blocked 10% of human IgG binding to represented in Fig. 6A. The results showed that CD32A has higher R CHO-CD32AH (Fig. 5A, compare top and bottom panel). Sim- binding affinity than CD32A for IgG1, IgG2, and IgG3 (Fig. 6B). ilar results were observed when EA was used as an IC (Fig. 5B). Both alleles have a similarly low-binding affinity for IgG4. We ob- These data suggest that the CD32AH-Ig cross-blocks the ligand served that CD32AH has 1.8-, 5.8-, and 2.22-fold higher affinity for binding mediated by cell surface CD32AR alleles, whereas IgG1, IgG2, and IgG3 when compared with CD32AR (Table I). For CD32AR-Ig is unable to cross-block the ligand binding medi- both CD32A alleles, the binding affinity for human IgG subtypes ated by cell surface CD32AH allele. ranked as IgG3ϾIgG1ϾIgG2ϾIgG4. These results indicate that the 8222 INTERACTION OF CD32A ALLELES WITH IMMUNE COMPLEXES

FIGURE 6. Representation of two-dimensional affinity measurement by micropipette method. The typical experimental setup is represented. A, Briefly, two glass micropipettes and the chamber were filled with binding buffer. A micropipette-aspirated RBC coated with human IgG subtypes was driven by a piezoelectric translator to make a 10-s contact with a CHO cell expressing CD32A alleles, held stationary by another pipette (cell contact). At the end of the contact duration, the two cells were separated by retracting the RBC. Upon retraction, an adhesion event between the two cells was indicated by stretched RBC membrane (cell binding). Nonspecific binding is determined by using RBC coated with BSA (no binding). B, CD32AH allele has a higher affinity for human IgG subtypes than the CD32AR allele as measured by micropipette method. The micropipette adhesion frequency assay was conducted as described in Materials and Methods. The contact test cycle as described in A was repeated 100 times using the same pair of cells, and the number of adhesion events was counted to obtain an adhesion frequency (Pa). The effective binding affinity (AcKa) was calculated by fitting the data into the equation described in Materials and Methods. Data are representative of two indi- p Ͻ ,ءء p Ͻ 0.01 and ,ء .vidual experiments 0.001.

CD32A alleles not only differ in human IgG2 binding, but also differ in protective immunity against bacterial infections (42, 43). In this substantially in their affinity for IgG1 and IgG3. report, we have shown that CD32A R and H alleles not only differ in their binding to human IgG2, which is in agreement with earlier Discussion reports, but also differ significantly in their affinity for IgG1 and The importance of low-affinity Fc␥R in the development of au- IgG3. These results suggest that reduced binding of CD32AR to toimmune diseases and defense against bacterial infections has IgG1 and IgG3 may also be a contributing factor to the higher recently been well documented (16, 22, 23, 25, 27). The con- incidence of bacterial infections in CD32AR homozygous indi- sequences of polymorphisms in CD32A, particularly their dif- viduals. Our observation with IgG1 binding contrasts but agrees ferent ligand binding characteristics, are being intensely inves- on IgG3 binding with a previous report by Bredius et al. (44) tigated due to their role in certain infectious and autoimmune that shows that CD32A alleles differ in binding to IgG2 and IgG3 diseases. CD32AH has been shown to bind to IgG2 with high but not IgG1. A recent report by Bruhns et al. (9) has shown that affinity, whereas CD32AR shows little or no binding to IgG2 CD32A alleles do not differ in binding to IgG1 and IgG3. At (16–18, 39–41). Several studies have documented that CD32AR ho- present, the reason for these discrepancies is not clear but it may mozygous individuals are more susceptible to bacterial infections be due to the use of different cell types and molecules. The report H R/H when compared with CD32A homozygous or CD32A heterozy- by Bredius et al. (44) used neutrophils treated with anti-CD16 gous individuals (7, 8). Most of the bacterial infections elicit predom- mAb, whereas Bruhns et al. (9) used CD32A fused with FLAG inantly IgG2 Ab response (16–18). Because CD32A is the only peptide either as expressed on CHO cells or purified molecules. In ␥ known Fc R that binds to IgG2 with high affinity, individuals ho- this report, we have used CHO cells expressing the unmodified R mozygous for CD32A might be more susceptible to bacterial infec- CD32A alleles and purified dimeric CD32A-Igs. Furthermore, we H tions when compared with CD32A homozygous individuals due to have used a two-dimensional affinity measurement technique (33), the lack of CD32A-IgG2 interaction. which directly measures the affinity of a receptor-ligand pair ex- In addition to the IgG2-mediated response, there are several pressed on cell membranes when they are mediating cell-cell ad- studies that document the pivotal role of IgG1 and IgG3 subtypes hesion as happens during Fc␥R-expressing cells binding to Ab- coated target cells. Interestingly, CD32AR/H heterozygous individuals are not sus- Table I. Affinity of cell surface CD32A alleles by two-dimensional affinity measurement ceptible to bacterial infections and have a phenotype similar to CD32AH individuals. To determine whether this effect is due to the H Affinity (␮m4) dominant effect of CD32A binding to IC, we studied the com- Human IgG petition between these alleles for ligand binding using purified Subtypes Human IgG1 Human IgG2 Human IgG3 Human IgG4 molecules. The results of the competition assay using rabbit IgG- CHO-CD32AR 7.98 ϫ 10Ϫ06 2.34 ϫ 10Ϫ06 1.45 ϫ 10Ϫ05 1.31 ϫ 10Ϫ06 coated SRBC suggest that when both alleles are exposed to the H Ϫ05 Ϫ05 Ϫ05 Ϫ06 CHO-CD32A 1.77 ϫ 10 1.38 ϫ 10 2.61 ϫ 10 1.39 ϫ 10 same ligand simultaneously, CD32AH outcompetes CD32AR for The Journal of Immunology 8223 ligand binding. Furthermore, our results show that purified References H CD32A -Ig is capable of blocking both the cell surface CD32A R 1. Hulett, M. D., and P. M. Hogarth. 1994. Molecular basis of Fc receptor function. and H alleles from binding to human IgG subtypes, whereas pu- Adv. Immunol. 57: 1–127. R 2. Kimberly, R. P., J. E. Salmon, and J. C. Edberg. 1995. Receptors for immuno- rified CD32A -Ig was unable to cross-block the binding mediated globulin G: molecular diversity and implications for disease. Arthritis Rheum. 38: H by cell surface CD32A allele. These differences in the ability to 306–314. compete for the ligand and cross-block each other may be due to 3. Ravetch, J. V. 1997. Fc receptors. Curr. Opin. Immunol. 9: 121–125. 4. Unkeless, J. C., E. Scigliano, and V. H. Freedman. 1988. Structure and function differences in the strength of the cell-cell adhesion mediated by of human and murine receptors for IgG. Annu. Rev. Immunol. 6: 251–281. these molecules, as evidenced by differences in their two-dimen- 5. Selvaraj, P., N. Fifadara, S. Nagarajan, A. Cimino, and G. Wang. 2004. Func- sional affinity. For instance, CD32AH has a 2-fold higher affinity tional regulation of human neutrophil Fc␥ receptors. Immunol. Res. 29: 219–230. 6. Selvaraj, P., W. F. Rosse, R. Silber, and T. A. Springer. 1988. The major Fc for IgG1 and IgG3 and a 5-fold higher affinity for IgG2 compared receptor in blood has a phosphatidylinositol anchor and is deficient in paroxysmal with the CD32AR allele. The results from binding and affinity stud- nocturnal hemoglobinuria. Nature 333: 565–567. ies suggest that individuals homozygous for the CD32AR allele 7. Parren, P. W., P. A. Warmerdam, L. C. Boeije, J. Arts, N. A. Westerdaal, A. Vlug, P. J. Capel, L. A. Aarden, and J. G. van de Winkel. 1992. On the have lower binding affinity for all human IgG subtypes and there- interaction of IgG subclasses with the low affinity Fc␥RIIa (CD32) on human fore may not be able to bind to Ab-coated bacteria effectively, monocytes, neutrophils, and platelets: analysis of a functional polymorphism to becoming susceptible to infection as a result. In individuals ho- human IgG2. J. Clin. Invest. 90: 1537–1546. H 8. Salmon, J. E., J. C. Edberg, N. Brogle, and R. P. Kimberly. 1992. Allelic Poly- mozygous for CD32A allele, the H allele has a higher affinity for morphisms of Human Fc␥ receptor IIA and Fc␥ receptor IIIB: independent mech- all human IgG subtypes and binds more effectively to Ab-coated anism for differences in human phagocyte function. J. Clin. Invest. 89: 1274–1281. bacteria leading to quicker clearance of infection. In heterozygous 9. Bruhns, P., B. Iannascoli, P. England, D. A. Mancardi, N. Fernandez, S. Jorieux, H individuals, as the CD32A allele has a higher affinity for all hu- and M. Dae¨ron. 2009. Specificity and affinity of human Fc␥ receptors and their man IgG subtypes, it is able to outcompete the CD32AR allele for polymorphic variants for human IgG subclasses. Blood 113: 3716–3725. 10. Sanders, L. A., J. G. van de Winkel, G. T. Rijkers, M. M. Voorhorst-Ogink, binding to Ab-coated bacteria. Consequently, the heterozygous in- M. de Haas, P. J. Capel, and B. J. Zegers. 1994. Fc␥ receptor IIa (CD32) heter- dividuals display a phenotype similar to the CD32AH homozygous ogeneity in patients with recurrent bacterial respiratory tract infections. J. Infect. individuals. Dis. 170: 854–861. 11. Yee, A. M., H. M. Phan, R. Zuniga, J. E. Salmon, and D. M. Musher. 2000. Apart from bacterial infections, studies have shown an associ- Association between Fc␥RIIa-R131 allotype and bacteremic pneumococcal pneu- ation between CD32AR and IC-mediated autoimmune diseases monia. Clin. Infect. Dis. 30: 25–28. 12. Domingo, P., E. Muniz-Diaz, M. A. Baraldes, M. Arilla, N. Barquet, R. Pericas, such as SLE (20, 21). In humans, individuals who express only ␥ R C. Juarez, P. Madoz, and G. Va´zquez. 2002. Association between Fc receptor CD32A are more susceptible to Ab-mediated autoimmune dis- IIa polymorphisms and the risk and prognosis of meningococcal disease. Am J eases than individuals expressing the CD32AH allele, which binds Med. 112: 19–25. to human IgG with higher affinity (16–18). This increased suscep- 13. Platonov, A. E., G. A. Shipulin, I. V. Vershinina, J. Dankert, J. G. van de Winkel, and E. J. Kuijper. 1998. Association of human Fc␥RIIa (CD32) polymorphism R tibility in CD32A individuals is suggested to be due to the in- with susceptibility to and severity of meningococcal disease. Clin. Infect. Dis. 27: ability of CD32AR to bind and clear IC from circulation leading to 746–750. 14. Moens, L., E. Van Hoeyveld, J. Vrhaegen, K. De Boeck, W. E. Peetermans, and the accumulation and deposition of IC on tissues (26, 27, 45, 46). X. Bossuyt. 2006. Fc␥ receptor IIA genotype and invasive pneumococcal infec- In humans, ICs formed are not only with IgG2 but also with other tion. Clin. Immunol. 118: 20–23. IgG subtypes and therefore the accumulation of IC in CD32AR 15. Endeman, H., M. C. Cornips, J. C. Grutters, J. M. van der Bosch, H. J. Ruven, H. van Velzen-Blad, G. T. Rijkers, and D. H. Biesma. 2009. The Fc␥ receptor individuals cannot be attributed to the inefficient binding of IgG2 IIA-R/R131 genotype is associated with sever sepsis in community acquired alone. Our findings suggest that CD32AR individuals have a lower pneumonia. Clin. Vaccine Immunol. 16: 1087–1090. affinity not only for IgG2 but also for human IgG1 and IgG3 sub- 16. van Sorge, N. M., W.-L. van der Pol, and J. G. J. Van de Winkel. 2003. Fc␥R polymorphisms: implications for function, disease susceptibility and immuno- types, and this might explain why IC from the circulation are not therapy. Tissue Antig. 61: 189–202. cleared efficiently. The IC deposited on tissues in CD32AR ho- 17. Sanders, L. A., R. G. Feldman, M. M. Voorhorst-Ogink, M. de Haas, mozygous individuals may lead to the development or acceleration G. T. Rijkers, P. J. Capel, B. J. Zegers, and J. G. van de Winkel. 1995. Human immunoglobulin G (IgG) Fc receptor IIA (CD32) polymorphism and IgG2-me- of the disease via complement activation and engagement of other diated bacterial phagocytosis by neutrophils. Pediatr. Infect. Dis. J. 63: 73–81. activating Fc␥Rs such as CD16. Several studies suggest the in- 18. Ferrante, A., L. J. Beard, and R. G. Feldman. 1990. IgG subclass distribution of volvement of complement proteins (47–52) and CD16 (16, 53, 54) antibodies to bacterial and viral antigens. Pediatr. Infect. Dis. J. 9: S16–SS24. 19. Yee, A. M. F., S. C. Ng, R. E. Sobel, and J. E. Salmon. 1997. Fc␥RIIA poly- in the development of autoimmune diseases. morphism as a risk factor for invasive pneumococcal infections in systemic lupus In conclusion, our results demonstrate that the H allelic form of erythematosus. Arthritis Rheum. 40: 1180–1182. 20. Karassa, F. B., T. A. Trikalions, and J. P. Ioannidis. 2002. Role of Fc␥ receptor CD32A has a higher affinity for human IgG1, IgG2, and IgG3 IIa polymorphism in susceptibility to systemic lupus erythematosus and lupus subtypes and outcompetes the R allelic form for ligand binding nephritis: a meta-analysis. Arthritis Rheum. 46: 1563–1571. when both alleles are coexpressed on the same cell surface. As a 21. Dijstelbloem, H. M., R. H. Scheepers, W. W. Oost, C. A. Stegeman, R/H W. L. van Der Pol, W. J. Sluiter, C. G. Kallenberg, J. G. van de Winkel, and result, the inflammatory cells from CD32A heterozygous indi- J. W. Tervaert. 1999. Fc␥ receptor polymorphisms in Wegener’s granulomatosis: viduals may predominantly use the H allele to mediate Ab-coated risk factors for disease relapse. Arthritis Rheum. 42: 1823–1827. target cell binding during phagocytosis and Ab-dependent cellular 22. Ludo van der Pol, W., and G. J. van de Winkel. 1998. IgG receptor polymor- H phism: risk factor for disease. Immunogen 48: 222–232. cytotoxicity, resulting in a phenotype similar to CD32A homozy- 23. Tan, S. Y. 2000. Fc␥RIIa polymorphism in systemic lupus erythematosus. Kidney gous individuals. Furthermore, our data also suggest that CD32AR Blood Press Res. 23: 138–142. has lower affinity not only for the IgG2 subtype but also for IgG1 24. Edberg, J. C., E. Wainstein, J. Wu, E. Csernok, M. C. Sneller, G. S. Hoffman, E. C. Keystone, W. L. Gross, and R. P. Kimberly. 1997. Analysis of Fc␥RII gene and IgG3 subtypes, which results in the lack of clearance of IC polymorphisms in Wegener’s granulomatosis. Exp. Clin. Immunogenet. 14: from circulation leading to IC-mediated autoimmune disease 183–195. susceptibility. 25. Manger, K., R. Repp, B. M. Spriewald, A. Rascu, A. Geiger, R. Wassmuth, N. A. Westerdaal, B. Wentz, B. Manger, J. R. Kalden, and J. G. van de Winkel. 1998. Fc␥ receptor IIa polymorphism in Caucasian patients with systemic lupus Acknowledgments erythromatosus: association with clinical symptoms. Arthritis Rheum. 41: 1181–1189. We thank Saranya Selvaraj, Archana Boopathy, Erica Bozeman, and Jaina 26. Carlsson, L. E., S. Satoso, G. Baurichter, H. Kroll, S. Papenberg, P. Eichler, Patel for critical reading of the manuscript. N. A. Westerdaal, V. Kiefel, J. G. van de Winkel, and A. Greinacher. 1998. Heparin-induced thrombocytopenia: new insights into the impact of the Fc␥RIIa- R-H131 polymorphims. Blood 92: 1526–1531. Disclosures 27. Dijstelbloem, H. M., M. Bijl, R. Fijnheer, R. H. Scheepers, W. W. Oost, The authors have no financial conflict of interest. M. D. Jansen, W. J. Sluiter, P. C. Limburg, R. H. Derksen, and 8224 INTERACTION OF CD32A ALLELES WITH IMMUNE COMPLEXES

J. G. van de Winkel. 2000. Fc␥ receptor polymorphisms in systemic lupus ery- 41. Warmerdam, P. A. M., J. G. J. VanDeWinkel, A. Vlug, N. A. C. Westerdaal, and thematosus: association with disease and in vivo clearance of immune complexes. P. J. A. Capel. 1991. A single amino acid in the second Ig-like domain of the Arthritis Rheum. 43: 2793–2800. human Fc-␥ receptor II is critical for human IgG2 binding. J. Immunol. 147: 28. Li, P., S. Nagarajan, C. Zhu, and P. Selvaraj. 2001. Recombinant CD16A-Ig 1338–1343. forms a homodimer and cross-blocks the ligand binding functions of neutrophil 42. Falconer, A. E., R. Carson, R. Johnstone, P. Bird, M. Kehoe, and J. E. Calvert. and monocyte Fc␥ receptors. Mol. Immunol. 38: 527–538. 1993. Distinct IgG1 and IgG3 subclass responses to two streptococcal protein 29. Chappel, M. S., D. E. Isenman, M. Everett, Y. Y. Xu, K. J. Dorrington, and antigens in man: analysis of antibodies to streptolysin O and M protein using M. H. Klein. 1991. Identification of the Fc␥ receptor class I binding site in human standardized subclass-specific enzyme-linked immunosorbent assays. Immunol- IgG through the use of recombinant IgG1/IgG2 hybrid and point-mutated anti- ogy 79: 89–94. bodies. Proc. Natl. Acad. Sci. USA 88: 9036–9040. 43. Hussain, R., A. Kifayet, and T. J. Chiang. 1995. Immunoglobulin G1 (IgG1) and 30. Chappel, M. S., D. E. Isenman, R. Oomen, Y. Y. Xu, and M. H. Klein. 1993. IgG3 antibodies are markers of progressive disease in leprosy. Pediatr. Infect. Identification of a secondary Fc␥RI binding site within a genetically engineered Dis. J. 63: 410–415. human IgG antibody. J. Biol. Chem. 268: 25124–25131. 44. Bredius, R. G., C. A. Fijen, M. de Haas, E. J. Kuijper, R. S. Weening, 31. Ledbetter, J. A., L. K. Gilliand, M. S. Hayden, P. S. Linsley, J. Bajorath, and J. G. van de Winkel, and T. A. Out. 1994. Role of neutrophil Fc␥RIIa (CD32) and H. P. Fell. 2000. Expression vectors encoding bispecific fusion proteins and Fc␥RIIIb (CD16) polymorphic forms in phagocytosis of human IgG1- and IgG3- methods of producing biologically active bispecific fusion proteins in mammalian opsonized bacteria and erythrocytes. Immunology 83: 624–630. cells. United States Patent Application Publication 08/539,436 Patent No 45. Miescher, S., M. O. Spycher, H. Amstutz, M. de Haas, M. Kleijer, U. J. Kalus, 6132992. H. Radtke, A. Hubsch, I. Andresen, R. M. Martin, and J. Bichler. 2004. A single 32. Shashidharamurthy, R., R. A. Hennigar, S. Fuchs, P. Palaniswami, M. Sherman, recombinant anti-RhD IgG prevents RhD immunization: association of RhD- and P. Selvaraj. 2008. Extravasations and emigration of neutrophils to the in- positive red blood cell clearance rate with polymorphisms in the Fc␥RIIA and flammatory site depend on the interaction of immune-complex with Fc␥ receptors Fc␥IIIA genes. Blood 103: 4028–4035. and can be effectively blocked by decoy Fc␥ receptors. Blood 111: 894–904. 46. Zuniga, R., G. S. Markowitz, T. Arkachaisri, E. A. Imperatore, V. D. D’Agati, 33. McHugh, R. S., S. N. Ahmed, Y.-C. Wang, K. W. Sell, and P. Selvaraj. 1995. and J. E. Salmon. 2003. Identification of IgG subclasses and C-reactive protein in Construction, purification and functional reconstitution on tumor cells of glyco- lupus nephritis: the relationship between the composition of immune deposits and lipid-anchored human B7-1 (CD80). Proc. Natl. Acad. Sci. USA 92: 8059–8063. Fc␥ receptor type IIA alleles. Arthritis Rheum. 48: 460–470. 34. Chesla, S. E., P. Selvaraj, and C. Zhu. 1998. Measuring two-dimensional recep- 47. Ingram, G., S. Hakobyan, N. P. Robertson, and B. P. Morgan. 2009. Complement tor-ligand binding kinetics by micropipette. Biophys. J. 75: 1553–1572. in multiple sclerosis: its role in disease and potential as biomarker. Clin. Exp. 35. Pricop, L., J. E. Salmon, J. C. Edberg, and A. J. Beavis. 1997. Flow cytometric Immunol. 155: 128–139. quantitation of attachment and phagocytosis in phenotypically-defined subpopu- 48. Lessey, E., N. Li, L. Diaz, and Z. Liu. 2008. Complement and cutaneous auto- lations of cells using PKH26-labeled Fc␥R-specific probes. J. Immunol. Methods immune blistering diseases. Immunol. Res. 41: 223–232. 205: 55–65. 49. Holers, V. M. 2008. The spectrum of complement alternative pathway-mediated 36. Williams, T. E., P. Selvaraj, and C. Zhu. 2000. Concurrent binding to multiple diseases. Immunol. Rev. 223: 300–316. ligands: kinetic rates of CD16b for membrane-bound IgG1 and IgG2. Biophys. J. 50. Ruiz-Arguelles, A., and L. Llorente. 2007. The role of complement regulatory 79: 1858–1866. proteins (CD55 and CD59) in the pathogenesis of autoimmune hemocytopenias. 37. Williams, T. E., S. Nagarajan, P. Selvaraj, and C. Zhu. 2000. Concurrent and Autoimmun. Rev. 6: 155–161. independent binding of Fc␥ receptors IIa and IIIb to surface-bound IgG. Biophys. 51. Seelen, M. A., and M. R. Daha. 2006. The role of complement in autoimmune J. 79: 1867–1875. renal disease. Autoimmunity 39: 411–415. 38. Shashidharamurthy, R., A. Amano, D. Ezekwudo, and P. Selvaraj. 2007. Analysis 52. Lemieux, R., and R. Bazin. 2006. Autoantibody-induced formation of immune of competitive interaction of H and R allelic forms of CD32A with rabbit IgG complexes in normal human serum. Curr. Pharm. Des. 12: 173–179. immune-complex. Proceedings of the 13th International Congress of Immunol- 53. Meyer, D., C. Schiller, J. Westermann, S. Izui, W. L. W. Hazenbos, J. S. Verbeek, ogy, August 21–25. Rio de Janeiro, p. 513. R. E. Schmidt, and J. E. Gessner. 1998. Fc␥RIII (CD16)-deficient mice show IgG 39. Clark, M. R., S. B. Clarkson, P. A. Ory, N. Stollman, and I. M. Goldstein. 1989. isotype-dependent protection to experimental autoimmune hemolytic anemia. Molecular basis for a polymorphism involving Fc receptor II on human mono- Blood 92: 3997–4002. cytes. J. Immunol. 143: 1731–1734. 54. Wu, J., J. C. Edberg, P. B. Redecha, V. Bansal, P. M. Guyre, K. Coleman, 40. Clark, M. R., S. Stuart, R. P. Kimberly, P. A. Ory, and I. M. Goldstein. 1991. A J. E. Salmon, and R. P. Kimberly. 1997. A novel polymorphism of Fc␥RIIIa single amino acid distinguishes the high-responder from the low-responder form (CD16) alters receptor function and predisposes to autoimmune disease. J. Clin. of Fc receptor II on human monocytes. Eur. J. Immunol. 21: 1911–1916. Invest. 100: 1059–1070.