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Comparative Study
. 2009 Dec 15;183(12):8216-24.
doi: 10.4049/jimmunol.0902550.

Dynamics of the interaction of human IgG subtype immune complexes with cells expressing R and H allelic forms of a low-affinity Fc gamma receptor CD32A

Rangaiah Shashidharamurthy  1 , Fang Zhang, Aaron Amano, Aparna Kamat, Ravichandran Panchanathan, Daniel Ezekwudo, Cheng Zhu, Periasamy Selvaraj
Affiliations
Comparative Study

Dynamics of the interaction of human IgG subtype immune complexes with cells expressing R and H allelic forms of a low-affinity Fc gamma receptor CD32A

Rangaiah Shashidharamurthy et al. J Immunol. .

Abstract

CD32A, the major phagocytic FcgammaR in humans, exhibits a polymorphism in the ligand binding domain. Individuals homozygous for the R allelic form of CD32A (CD32A(R) allele) are more susceptible to bacterial infections and autoimmune diseases as compared with H allelic CD32A (CD32A(H)) homozygous and CD32A(R/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 CD32A(H)-Ig outcompetes CD32A(R)-Ig for IC binding when both alleles simultaneously compete for the same ligand. CD32A(H)-Ig blocked the IC binding mediated by both the allelic forms of cell surface CD32A, whereas CD32A(R)-Ig blocked only CD32A(R) and was unable to cross-block IC binding mediated by CD32A(H). Two-dimensional affinity measurements also demonstrated that CD32A(R) has significantly lower affinity toward all three subtypes as compared with CD32A(H). Our data suggest that the lower binding of CD32A(R) 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 CD32A(R/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 CD32A(H) homozygous individuals.

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Figures

Figure 1
Figure 1. Detection of CD32A-Ig dimers
SDS-PAGE analysis of immunoaffinity purified dimeric CD32AR-Ig (left lane) and CD32AH-Ig (right lane) alleles. Dimers were purified using Gamma bind plus column from CHO cell culture supernatant. The purified protein (5μg) was subjected to SDS-PAGE under reducing (left panel) and non-reducing (right panel) conditions and the protein bands were visualized using the silver staining (A) method and Western blotting (B).
Figure 2
Figure 2. Binding of CD32A-Ig molecules to soluble immune complex (sIC)
A 96 well plate was coated with 50 μl (10 μg/ml) CD32A-Ig molecules overnight at 4°C and wells were blocked with binding buffer (PBS/5 mM EDTA with 1% BSA). After washing the wells three times with binding buffer, 50 μl of HRP conjugated sIC of human IgG subtypes (5 μg/ml) were added and incubated for another 1h at 4 °C. Wells were washed three times; color was developed by adding the HRP substrate and read at 450 nm. IV.3 treated wells served as a specificity control. The ELISA O.D readings after blocking with IV.3 mAb were as follows. For CD32AH-Ig coated wells: IgG1=0.017, IgG2=0.019, IgG3=0.016, IgG4=0.018, and for CD32AR-Ig coated wells: IgG1=0.011, IgG2=0.015, IgG3=0.017, IgG4=0.02. The values are average of triplicate readings and representative of three individual experiments. The values are comparable with BSA coated negative control wells. *p<0.01, **P<0.001.
Figure 3
Figure 3
(A) 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). Open histogram shows the isotype control, solid histogram indicates the binding of IV.3 antibody to CD32A alleles. Dotted line indicates the expression level of CD32A alleles. (B) Binding of cell surface CD32A alleles to soluble immune complex (sIC) of human IgG subtypes. CHO cells expressing CD32A alleles were incubated with FITC conjugated sIC of human IgG subtypes (10 μg/ml) in the presence and absence of IV.3 (10 μg/ml) and analyzed for FITC-IC binding to CHO-CD32A alleles using flow cytometry (solid histogram). IV.3 treated cells served as a negative control (open histogram). Data is representative of three individual experiments. (C). Graphical representation of human IgG subtypes sIC bound to CHO-CD32A alleles. Values are mean ± SD of data from three experiments. *p<0.01, **P<0.001. (D) Binding of cell surface 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 μl of 1.5 x 108 cells) for 2 h at 4°C. Values are mean ± SD of data from three experiments. EA bound to the cells were 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 following formula: % cells bound to EA x mean fluorescence/100. Values are mean ± SD of data from three experiments. *p<0.01, **P<0.001.
Figure 4
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 μl of EA (2 × 108 cells/ml) for 1h at 4 °C. After washing three times with binding buffer, the incubation was continued with F(ab′)2 of goat anti human Fc specific antibody conjugated with FITC for 1 h at 4 °C. Then 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 were mixed and incubated with EA. The CD32A-Ig molecules were preincubated with F(ab′)2 of goat anti human Fc specific antibody conjugated with Cy5 (for CD32AH-Ig) or FITC (for CD32AR-Ig) for 1 h at 4 °C and then 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 antibody alone served as a specificity control. (I) EA treated with secondary antibody alone, (II) EA incubated with 50 μg/ml of FITC-CD32AR-Ig, (III) EA incubated with 50 μg/ml of Cy5-CD32AH-Ig, (IV) EA incubated with CD32AR-Ig (50 μg/ml) and CD32AH-Ig (25 μg/ml), (V) EA incubated with CD32AR-Ig (25 μg/ml) and CD32AH-Ig (25 μg/ml), (VI) EA incubated with CD32AR-Ig (50 μg/ml) and CD32AH-Ig (50 μg/ml). Lower left quadrant: unstained cells, lower right: CD32AR-Ig bound EA, upper left: CD32AH-Ig bound EA, upper right: EA bound to both the CD32A-Ig molecules. Data is representative of three individual experiments.
Figure 5
Figure 5
(A) Soluble CD32A-Ig molecules compete with CD32A alleles expressed on CHO cell surface and block binding of soluble immune-complex of human IgG subtypes. A: CHO-CD32AR (upper panel) or CHO-CD32AH (lower panel) cells were incubated with FITC-IC (sIC; 10 μg/ml) in the presence and absence of CD32A-Ig molecules (50 μg/ml) and analyzed. IV.3 antibody treated cells served as a specificity control. Blocking reagents are represented on the Y-axis. Values are mean ± SD of data from three experiments. *p<0.01, **P<0.001. (B) Soluble CD32A-Ig molecules compete with cell surface CHO-CD32A alleles and block binding of erythrocytes coated with human IgG subtypes. CHO-CD32AR (upper panel) or CHO-CD32AH (lower panel) cells were incubated human IgG subtype coated, PKH-labeled-EA (50 μl of 1.5 × 108 cells) for 2 h at 4 °C in the presence and absence of purified CD32A-Ig molecules (50 μg/ml). The cells were analyzed for binding of EA using flow cytometry. IV.3 treated cells and cells incubated with PKH-labeled unopsonized SRBCs served as a specificity control. Blocking reagents are represented on the Y-axis. The binding index was calculated using the following formula: % cells bound to EA × mean fluorescence/100. Values are mean ± SD of data from three experiments. *p<0.01, **P<0.001.
Figure 6
Figure 6
(A) Typical picture of 2D Affinity measurement by micropipette method The typical experimental setup is represented in this figure. Briefly, two glass micropipettes and the chamber medium were filled with binding buffer. A micropipette-aspirated red blood cell coated with human IgG subtypes was driven by a piezoelectric translator to make a 10 second 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 red blood cell. Upon retraction, an adhesion event between the two cells was indicated by stretched red blood cell membrane (cell binding). Nonspecific binding is determined by using red blood cells 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 carried out as described under Materials and Methods. The contact test cycle as described in figure 6A was repeated 100 times using the same pair of cells, keeping the contact duration (t) and the area (Ac, ~3 μm−2) constant, and the number of adhesion events was counted to obtain an adhesion frequency (Pa). The effective binding affinity (AcKa) and off-rate (kr) were extracted by fitting the data into the equation as described under Materials and Methods. Data is representative of two individual experiments. *p<0.01, **P<0.001.
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