Molecular and Functional Characterization of Fcγ Receptor IIIb-Ligand Interaction: Implications for Neutrophil-Mediated Immune Mechanisms in Malaria

Abstract

The Fcγ receptor IIIb (FcγRIIIb) is a low-affinity receptor of IgG and is essential in neutrophil-mediated effector functions. Different allelic forms of FcγRIIIb carrying human neutrophil antigen (HNA-1a, -1b, -1c, and -1d) have been identified. Here, we have generated stable transfected HEK293 cell lines expressing HNA-1aa, -1bb, and -1bc. Of these, cells expressing HNA-1bc interacted significantly stronger (binding affinities, 2.277 versus 0.743) with human IgG than cells expressing the HNA-1aa or -1bb alloforms. The higher affinity of IgG toward the HNA-1c alloform was confirmed using neutrophils derived from German blood donors. Neutrophils from HNA-1abc-phenotyped individuals bound IgG significantly stronger (1.825 versus 0.903) than did neutrophils from HNA-1ab-typed individuals. These findings were confirmed by surface plasmon resonance (SPR) analysis demonstrating that recombinant HNA-1bc had a higher affinity (dissociation constant [K_d ], 7.24 × 10^-6 M) than recombinant HNA-1bb (K_d , 1.15 × 10^-5 M) against normal IgG. Finally, we demonstrated that Plasmodium falciparum merozoites opsonized with human IgG affinity purified against P. falciparum glutamate-rich protein (GLURP) enhanced stronger reactive oxygen species (ROS) emission in neutrophils obtained from HNA-1abc donors than in neutrophils from HNA-1ab donors. Collectively, these results indicate that the amino acid substitution Ala₇₈Asp resulting in the HNA-1c allotype leads to higher affinity toward human IgG, enhancement of neutrophil activation, and possibly effective clearance of malaria by intracellular ROS.

Keywords: IgG binding; alloantibodies; human neutrophil antigen-1; malaria.

FIG 1

Analysis of allelic forms of FcγRIIIb expressed on the surface of HEK293 transfected cells by flow cytometry and antigen capture assay (MAIGA). (A) Transfected HEK293 cells expressing HNA-1aa, -1bb, and -1bc were incubated with MAb LNK16 against FcγRIIIb as indicated. Mouse IgG was run as an isotype control. After washings, cells were labeled with fluorescence-conjugated donkey anti-mouse IgG and analyzed by flow cytometry. (B) Transfected cells expressing HNA-1aa, -1bb, and -1bc were incubated with anti-HNA-1a, -1b, and -1c aabs together with MAb LNK16. After lysis, a trimolecular complex consisting of the target protein, the capture MAb, and the alloantibody was immobilized onto a microtiter plate. Human antibodies bound to immobilized FcγRIIIb alloform were detected with enzyme-labeled goat anti mouse-IgG and read on an ELISA reader (optical density [OD] at 492/620 nm). Dashed lines are cutoff values for a positive result calculated as twice the OD values obtained with the normal control serum. Data are presented as means ± standard deviations (SD) for triplicates.

FIG 2

Adhesion of transfected HEK293 cells and neutrophils to immobilized IgG. (A) HEK cells. Adhesion of transfected HEK293 cells expressing HNA-1aa, -1bb, and -1bc antigens to IgGs. Transfected cells (1 × 10⁶ to 4 × 10⁶ cells) were allowed to adhere onto microtiter wells coated either with 5 μg IgG or with bovine serum albumin (BSA) for 1 h at 37°C in the presence of 5% CO₂. In some experiments, 3G8 or DJ130c MAb was added for 30 min at 37°C. After washings, bound cells were stained with crystal violet and their numbers were determined by a microplate reader. Data are normalized with BSA and adjusted to FcγRIIIb expression. Data are presented as means ± SD from five independent experiments. (B) Granulocytes. HNA-phenotyped granulocytes expressing HNA-1ab (n = 3), -1ac (n = 1), -1bc (n = 1), and -1abc (n = 3) antigens to IgGs. Neutrophils (1× 10⁶ to 4 × 10⁶ cells) were allowed to adhere onto microtiter wells coated either with 5 μg IgG or with bovine serum albumin (BSA) for 1 h at 37°C in the presence of 5% CO₂. After washings, bound cells were stained with crystal violet and their numbers were determined in an ELISA reader. Data are normalized with BSA and adjusted to FcγRIIIb expression. Data are presented as means ± SD.

FIG 3

Binding of FcγRIIIb alloforms onto IgG by ELISA and SPR. (A) ELISA. Biotin-labeled recombinant FcγRIIIb alloforms (HNA-1aa, -1bb, and -1bc) were added into microtiter wells coated with 2.5 μg IgG or BSA for 1 h at room temperature. After washings, bound FcγRIIIb protein was detected with HRP-conjugated streptavidin using TMB as the substrate. The color reaction was read on an ELISA reader at 450 nm. Data are presented as means ± SD from three independent experiments. (B) SPR. Different amounts of IgG fractions (50, 100, 200, 400, and 800 nmol/liter) were injected over three flow cells coated with different recombinant FcγRIIIb alloforms (HNA-1aa, -1bb, and -1bc). The binding response in real time was recorded as resonance units (response units) for 500 s. The dissociation constant (K_d) was analyzed using computer software (ProteOn Manager; Bio-Rad).

FIG 4

Model of the three-dimensional structure of FcγRIIIb alloforms (green) and the Fc part of IgG (blue) around their interface (blue surface). (a) Structure of the interface between the HNA-1b alloform of soluble FcγRIIIb and Fc [“proline sandwich”: Trp108, Pro212 (Fc), and Trp131] as presented by the crystal structure (Protein Data Bank accession code 1E4K [14]). (b) Model of the structure of the HNA-1c alloform, obtained by in silico mutation 78Ala>Asp. Additional polar interactions are possible, exemplified by a potential salt bridge between receptor residues Asp78 and Lys209 on the Fc side. (c and d) Model of the geometric arrangement of the two Ig-like C2-type domains of the HNA-1b alloform of FcγRIIIb (c) and of the HNA-1a alloform (d). The contact surface of domain 2 is in green.

FIG 5

ROS emission of HNA-1 phenotypes neutrophils primed with antibodies, in the presence of merozoites. HNA-1-phenotyped neutrophils derived from HNA-1ab (n = 4) and HNA-1abc (n = 4) donors were incubated with IgG (A) or specific IgG antibodies against malaria (B) in the presence of merozoites (see Materials and Methods). ROS emission derived from activated neutrophils was measured with DHR fluorescence probe by flow cytometry. The ratio between ROS with IgG mediation and without IgG (DHR alone) was calculated. Data are presented as means ± SD.