Fc gammaRIIIb triggers raft-dependent calcium influx in IgG-mediated responses in human neutrophils

Abstract

Human neutrophils constitutively express a unique combination of FcγRs, namely FcγRIIa and FcγRIIIb. Numerous lines of evidence support the concept that these FcγRs generate only partially characterized intracellular signals. However, despite the fact that both receptors are likely to be engaged simultaneously in a physiological setting, no recent publications have investigated the distinct, although partially convergent, results of their joint activation in IgG-dependent responses. To examine the significance of the co-expression of FcγRIIa and FcγRIIIb on human neutrophils, we analyzed the neutrophil responses to stimuli that engage these FcγRs, namely the phagocytosis of human IgG-opsonized zymosan and the responses to heat-aggregated IgGs. Blocking antibodies to either FcγR significantly decreased the phagocytic index and the stimulated production of superoxide anions. Both receptors are required for optimal IgG-dependent responses by human neutrophils. On the other hand, only blocking antibodies to FcγRIIIb, but not to FcγRIIa, inhibited the mobilization of calcium in response to heat-aggregated IgGs. Furthermore, phagocytosis of IgG-opsonized zymosan by human neutrophils required an extracellular influx of calcium that was blocked only by antibodies against FcγRIIIb. We also observed that this calcium influx as well as the IgG-dependent phagocytosis were dependent on the integrity of the plasma membrane detergent-resistant microdomains to which both isoforms were recruited following stimulation by heat-aggregated IgGs. These data clarify the mechanisms that regulate the FcγRs constitutively expressed on human neutrophils, describe a specific contribution of FcγRIIIb at the level of the mobilization of calcium, and provide evidence for a crucial role of detergent-resistant microdomains in this process.

FIGURE 1.

Both FcγRs isoforms are required in IgG-dependent phagocytosis and superoxide production. A and B, neutrophils were preincubated with the blocking monoclonal antibodies IV.3 F(ab′)₂ or 3G8 F(ab′)₂ for 5 min at 37 °C and incubated with fluorescent IgG-opsonized zymosan (ZO, A) or 25 μg of fluorescent HA-IgG (B) as described under “Experimental Procedures.” Fluorescence was measured by flow cytometry. These graphs are quantifications of the means of at least three independent phagocytosis experiments. C and D, neutrophils were preincubated with the blocking monoclonal antibodies IV.3 F(ab′)₂ or 3G8 F(ab′)₂ for 5 min at 37 °C, and cytochrome c (125 μm) was added. Superoxide production measurement was then initiated by the addition of IgG-opsonized zymosan (C) or HA-IgG (1 mg/ml) (D), and the reactions were stopped on ice after 10 min. The data shown are compiled from six independent experiments.

FIGURE 2.

Both FcγR isoforms are required in IgG-mediated signaling. Neutrophils were preincubated with monoclonal antibodies IV.3 F(ab′)₂ (1 μg/ml) or 3G8 F(ab′)₂ (4 μg/ml) for 5 min at 37 °C and stimulated with HA-IgG (1 mg/ml) for the indicated times. Whole cell lysates were loaded on the same gel and probed by immunoblotting for tyrosine phosphorylated residues (pY) (A) and FcγRIIa (CT10 antibody) (B and C). Ubiquitin ligase c-Cbl was probed as loading control. Compilations of densitometric ratios of four independent experiments are represented on the graphs. WB, Western blot; MW, molecular mass.

FIGURE 3.

FcγRIIIb mediates an influx of calcium in response to HA-IgG. Neutrophils were incubated with Fura-2-AM as described under “Experimental Procedures.” Monoclonal antibodies IV.3 F(ab′)₂ (1 μg/ml) or 3G8 F(ab′)₂ (4 μg/ml) were added for 5 min at 37 °C before stimulation. A, calcium mobilization was measured following the addition of HA-IgG (1 mg/ml). B and C, Mn²⁺ influx, monitored by the quenching of intracellular Fura-2, was measured following stimulation initiated with HA-IgG (1 mg/ml) (B) or anti-mouse F(ab′)₂ (C) as described under “Experimental Procedures.” These data are representative of five independent determinations. Ctrl, control.

FIGURE 4.

FcγR-dependent phagocytosis requires an influx of calcium. Neutrophils, in the presence or absence of 2 mm EGTA, were incubated with fluorescent IgG-opsonized zymosan as described under “Experimental Procedures.” Fluorescence was measured by flow cytometry. Phagocytic index (A) was calculated by multiplying the number of phagocytic cells (B) by the number of internalized zymosan per cell (mean fluorescence intensity, MFI) (C). These graphs are a quantification of the means of six independent experiments. Ctrl, control.

FIGURE 5.

DRMs integrity is essential for optimal FcγR-dependent responses. Neutrophils were preincubated or not with 10 mm mβCD for 15 min at 37 °C. A, neutrophils were incubated with fluorescent zymosan as described under “Experimental Procedures.” Fluorescence was measured by flow cytometry. For cholesterol repletion, neutrophils were washed after the mβCD incubation, and then 4 mm of cholesterol was added for 30 min at 37 °C and washed before the addition of opsonized zymosan. This graph is a quantification of the means of three independent experiments. B and C, neutrophils were incubated with Fura-2-AM as described under “Experimental Procedures,” and mβCD was added for the last 15 min. B and C, calcium influx (B) and calcium mobilization (C) were measured following HA-IgG (1 mg/ml) stimulation. These data are representative of four independent determinations. D and E, neutrophils were stimulated with HA-IgG (1 mg/ml) for the indicated times. Whole cell lysates were probed by immunoblotting for tyrosine phosphorylated residues (pY) (D) and FcγRIIa (E). Ubiquitin ligase c-Cbl was probed as loading control. Compilations of densitometric ratios of four independent experiments are represented on the graph. WB, Western blot; MW, molecular mass.

FIGURE 6.

FcγRIIa and FcγRIIIb co-fractionate in DRM_H upon FcγRs engagement. Neutrophils (40 × 10⁶ cells/ml) were preincubated with 1 mm DFP for 10 min at room temperature before stimulation at 37 °C for 30 s. Plasma membranes were prepared as described under “Experimental Procedures” and solubilized in 1% Nonidet P-40. A and B, solubilized plasma membranes from HA-IgG-stimulated neutrophils were subjected to ultracentrifugation at 100,000 × g on OptiPrep cushion. An aliquot of the pellet (insoluble membrane fraction) was probed for FcγRIIa (A, upper panels) and FcγRIIIb (B, upper panels). Insoluble membrane fraction from HA-IgG-stimulated neutrophils was fractionated on Optiprep gradient as described under “Experimental Procedures.” The gradient fractions were collected, and the proteins were precipitated and analyzed by immunoblotting for FcγRIIa (A, lower panel) and FcγRIIIb (B, lower panel). These data are representative of three independent experiments. C, FcγRIIa or FcγRIIIb were independently cross-linked on neutrophils using monoclonal antibodies as described under “Experimental Procedures.” Plasma membranes were isolated, solubilized, and ultracentrifuged. The two resulting independently obtained insoluble membrane fractions were then mixed before fractionation on Optiprep gradients. The fractions were analyzed by immunoblotting for FcγRIIa and FcγRIIIb. WB, Western blot.