The Antibody Receptor Fc Gamma Receptor IIIb Induces Calcium Entry via Transient Receptor Potential Melastatin 2 in Human Neutrophils

Abstract

Human neutrophils express two unique antibody receptors for IgG, the FcγRIIa and the FcγRIIIb. FcγRIIa contains an immunoreceptor tyrosine-based activation motif (ITAM) sequence within its cytoplasmic tail, which is important for initiating signaling. In contrast, FcγRIIIb is a glycosylphosphatidylinositol (GPI)-linked receptor with no cytoplasmic tail. Although, the initial signaling mechanism for FcγRIIIb remains unknown, it is clear that both receptors are capable of initiating distinct neutrophil cellular functions. For example, FcγRIIa is known to induce an increase in L-selectin expression and efficient phagocytosis, while FcγRIIIb does not promote these responses. In contrast, FcγRIIIb has been reported to induce actin polymerization, activation of β1 integrins, and formation of neutrophils extracellular traps (NET) much more efficiently than FcγRIIa. Another function where these receptors seem to act differently is the increase of cytoplasmic calcium concentration. It has been known for a long time that FcγRIIa induces production of inositol triphosphate (IP₃) to release calcium from intracellular stores, while FcγRIIIb does not use this phospholipid. Thus, the mechanism for FcγRIIIb-mediated calcium rise remains unknown. Transient Receptor Potential Melastatin 2 (TRPM2) is a calcium permeable channel expressed in many cell types including vascular smooth cells, endothelial cells and leukocytes. TRPM2 can be activated by protein kinase C (PKC) and by oxidative stress. Because we previously found that FcγRIIIb stimulation leading to NET formation involves PKC activation and reactive oxygen species (ROS) production, in this report we explored whether TRPM2 is activated via FcγRIIIb and mediates calcium rise in human neutrophils. Calcium rise was monitored after Fcγ receptors were stimulated by specific monoclonal antibodies in Fura-2-loaded neutrophils. The bacterial peptide fMLF and FcγRIIa induced a calcium rise coming initially from internal pools. In contrast, FcγRIIIb caused a calcium rise by inducing calcium entry from the extracellular medium. In addition, in the presence of 2-aminoethoxydiphenyl borate (2-APB) or of clotrimazole, two inhibitors of TRPM2, FcγRIIIb-induced calcium rise was blocked. fMLF- or FcγRIIa-induced calcium rise was not affected by these inhibitors. These data suggest for the first time that FcγRIIIb aggregation activates TRPM2, to induce an increase in cytoplasmic calcium concentration through calcium internalization in human neutrophils.

Keywords: Fc gamma receptor; PKC (protein kinase C); TRPM2 cation channel; calcium; neutrophil; reactive oxygen species.

Figure 1

Fcγ receptors induce an increase in [Ca²⁺]_i. (A–D) Fura-2-loaded human neutrophils in PBS with Ca²⁺ and Mg²⁺ were stimulated with 10 nM fMLF (A), or were stimulated by aggregating FcγRIIa on the cell membrane with mAb IV.3 (B), or by aggregating FcγRIIIb on the cell membrane with mAb 3G8 (C). (E–H) Fura-2-loaded neutrophils were resuspended in PBS containing 1 mM of EGTA and fluorescence changes detected after stimulating with 10 nM fMLF (E), or by aggregating FcγRIIa (F), or by aggregating FcγRIIIb (G). After 300 seconds, 4 mM Ca²⁺ was added and measurements continued until 450 seconds. Arrow indicates the moment when the stimulus was added. Changes in cytosolic calcium concentration ([Ca²⁺]_i) were assessed by measuring the variations in fluorescence. Tracings are representative of three experiments with similar results. (D) Increments in [Ca²⁺]_i were calculated by subtracting the baseline value from the maximum value after stimulation. (H) The initial rise in [Ca²⁺]_i represents Ca²⁺ from intracellular stores, while the rise in [Ca²⁺]_i after addition of 4 mM Ca²⁺ represents Ca²⁺ from extracellular medium. Data are mean ± SEM of three independent experiments. Asterisks denote conditions that were statistically different from untreated cells (p < 0.01).

Figure 2

Fcγ receptor-mediated increase in [Ca²⁺]_i is independent of TAK1 and MEK. Fura-2-loaded human neutrophils in PBS with Ca²⁺ and Mg²⁺ were left untreated (green line), or treated with 10 nM LLZ 1640-2 (LLZ) a TAK1 inhibitor, or with 50 µM UO126, a MEK inhibitor, or with 2 μg/ml Pertussis toxin (P Tox), before being stimulated by aggregating FcγRIIa (A), or by aggregating FcγRIIIb (B), or with 10 nM fMLF (C). Arrow indicates the moment when the stimulus was added. Changes in cytosolic calcium concentration ([Ca²⁺]_i) were assessed by measuring the variations in fluorescence as described in material and methods. Tracings are representative of three experiments with similar results. (D) Increments in [Ca²⁺]_i were calculated by subtracting the baseline value from the maximum value after stimulation. Data are mean ± SEM of three independent experiments. Asterisks (*) denote conditions that were statistically different from untreated cells (p < 0.01).

Figure 3

FcγRIIIb-mediated increase in [Ca²⁺]_i. depends on Syk, PKC and NADPH-oxidase. Fura-2-loaded human neutrophils in PBS with Ca²⁺ and Mg²⁺ were left untreated (green line), or treated with 1 µM iSyk, a Syk inhibitor; 1 µM Gö6976, a PKC inhibitor; 10 µM DPI, a NADPH-oxidase inhibitor, before being stimulated by aggregating FcγRIIa (A) or FcγRIIIb (B). Arrow indicates the moment when the stimulus was added. Changes in cytosolic calcium concentration ([Ca²⁺]_i) were assessed by measuring the variations in fluorescence as described in material and methods. Tracings are representative of three experiments with similar results. (C) Increments in [Ca²⁺]_i were calculated by subtracting the baseline value from the maximum value after stimulation. Data are mean ± SEM of three independent experiments. Asterisks (*) denote conditions that were statistically different from untreated cells (p < 0.0001).

Figure 4

FcγRIIIb-induced ROS production involves Syk and PKC. Reactive Oxygen Species (ROS) production was assessed in dihydrorhodamine 123-loaded neutrophils by detecting fluoresce changes in flow cytometry. (Upper part) Neutrophils were left untreated (light gray), or were stimulated (white) with 20 nM PMA, or by aggregating FcγRIIa, or by aggregating FcγRIIIb. Some neutrophils were previously treated (dark gray) with 1 µM iSyk, a Syk inhibitor; or 1 µM Gö6976, a PKC inhibitor; before being stimulated. (Lower part) Cumulative data (mean ± SEM) of mean fluorescence intensity (MFI) from three independent experiments done in triplicate. Asterisk (*) denote condition that statistically different from untreated cells (p < 0.001).

Figure 5

TRPM 2 channel mediates FcγRIIIb-induced increase in [Ca²⁺]_i. Fura-2-loaded human neutrophils in PBS with Ca²⁺ and Mg²⁺ were left untreated (blue line), or treated (red line) with 5 µM 2-APB (A) or with 10 µM clotrimazole (B), two TRPM2 inhibitors, before being stimulated by aggregating FcγRIIa or FcγRIIIb. Changes in cytosolic calcium concentration ([Ca²⁺]_i) were assessed by measuring the variations in fluorescence as described in material and methods. Tracings are representative of three experiments with similar results. (C) Increments in [Ca²⁺]_i were calculated by subtracting the baseline value from the maximum value after stimulation. Data are mean ± SEM of three independent experiments. Asterisks (*) denote conditions that were statistically different from untreated cells (p < 0.0008).

Figure 6

TRPM2 is not required for FcγRIIIb-induced ROS production. Reactive Oxygen Species (ROS) production was assessed in dihydrorhodamine 123-loaded neutrophils by detecting fluoresce changes by flow cytometry. Neutrophils were previously left untreated (—), or were treated with the 5 µM 2-APB, a TRPM2 inhibitor, before being stimulated with 20 nM PMA, or by aggregating FcγRIIa, or by aggregating FcγRIIIb. Data are mean ± SEM of mean fluorescence intensity (MFI) from three independent experiments done in triplicates.

Figure 7

Model for FcγRIIIb-mediated increase in [Ca²⁺]_i in human neutrophils (PMN). After aggregation of FcγRIIIb by antibody (Ab)/antigen (Ag) immune complexes, on the plasma membrane of neutrophils (PMN), spleen tyrosine kinase (Syk) gets activated, leading to protein kinase C (PKC) activation. PKC is then required for nicotinamide adenine dinucleotide phosphate oxidase (NADPH-oxidase) activation. NADPH-oxidase, an enzymatic complex assembled on a membrane (not shown), in turn produces reactive oxygen species (ROS), which induce adenosine diphosphate ribose (ADPR) finally leading to activation of transient receptor potential melastatin 2 (TRPM2) channels on the plasma membrane. Activated TRMP2 allow influx of extracellular Ca²⁺ into the cell. iSyk, an inhibitor of Syk; Gö6976, an inhibitor of PKC; DPI, an inhibitor of NADPH-oxidase; 2-APB, an inhibitor of TRPM2; Clotrimazole, another inhibitor of TRPM2.