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

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.

Human neutrophils express two unique antibody receptors for IgG, the FcgRIIa and the FcgRIIIb. FcgRIIa contains an immunoreceptor tyrosine-based activation motif (ITAM) sequence within its cytoplasmic tail, which is important for initiating signaling. In contrast, FcgRIIIb is a glycosylphosphatidylinositol (GPI)-linked receptor with no cytoplasmic tail. Although, the initial signaling mechanism for FcgRIIIb remains unknown, it is clear that both receptors are capable of initiating distinct neutrophil cellular functions. For example, FcgRIIa is known to induce an increase in L-selectin expression and efficient phagocytosis, while FcgRIIIb does not promote these responses. In contrast, FcgRIIIb has been reported to induce actin polymerization, activation of b1 integrins, and formation of neutrophils extracellular traps (NET) much more efficiently than FcgRIIa. 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 FcgRIIa induces production of inositol triphosphate (IP 3 ) to release calcium from intracellular stores, while FcgRIIIb does not use this phospholipid. Thus, the mechanism for FcgRIIIb-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 FcgRIIIb stimulation leading to NET formation involves PKC activation and reactive oxygen species (ROS) production, in this report we explored whether TRPM2 is activated via FcgRIIIb and mediates calcium rise in human neutrophils. Calcium rise was monitored after Fcg receptors were stimulated by specific monoclonal antibodies in Fura-2-loaded neutrophils. The bacterial peptide fMLF and FcgRIIa induced a calcium rise coming initially from internal pools. In contrast, FcgRIIIb 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, FcgRIIIb-induced calcium rise was blocked. fMLF-or FcgRIIa-induced calcium rise was not affected by these inhibitors. These data suggest for the first time that FcgRIIIb INTRODUCTION Neutrophils, the most abundant leukocytes in peripheral blood, are considered the first line of defense because these cells arrive first at sites of inflammation or infection (1,2). Once there, neutrophils display a variety of antimicrobial functions including phagocytosis (3,4), degranulation, and formation of neutrophil extracellular traps (NET) (5). These functions can be initiated or enhanced by antibodies, in the form of immune complexes, binding to their cognate Fc receptors on the surface of the neutrophil (6). Human neutrophils express constitutively two Fc receptors for IgG, the FcgRIIa (CD32a) and the FcgRIIIb (CD16b). FcgRIIa contains an ITAM (immunoreceptor tyrosinebased activation motif) sequence in its cytoplasmic tail (7), while FcgRIIIb is a glycosylphosphatidylinositol (GPI)-linked receptor, lacking a cytoplasmic tail (8). These two neutrophil antibody receptors are not only structurally different but also have been shown to induce unique cellular responses (9). FcgRIIa is the predominant Fcg receptor mediating phagocytosis, while the contribution of FcgRIIIb to this response is minimal (10). In contrast, FcgRIIIb signaling to the neutrophil nucleus for nuclear factor activation is more efficient than FcgRIIa signaling (11). In addition, FcgRIIIb is the only Fcg receptor capable of inducing NET formation (12,13). Although, these reports indicate that each receptor can activate particular cell responses, the signaling capabilities of each receptor are still incompletely understood.
Early reports clearly showed that both neutrophil Fcg receptors induce an increase in cytoplasmic Ca 2+ concentration ([Ca 2+ ] i ) (14,15). However, the mechanism for this response seems to be different for each receptor (15). FcgRIIa signals via its ITAM sequence to activate Syk (spleen tyrosine kinase). Syk then phosphorylates enzymes such as PI 3-K (phosphatidylinositol 3kinase) and PLCg (phospholipase Cg). PLCg produces inositoltriphosphate (IP 3 ), and diacylglycerol (DAG). These second messengers cause calcium release from the endoplasmic reticulum (ER), and activation of PKC (protein kinase C), respectively (8,16,17). In sharp contrast, FcgRIIIb-mediated increase in [Ca 2+ ] i does not involve IP 3 formation (15), and the mechanism used by FcgRIIIb to increase [Ca 2+ ] i is still unknown. Because FcgRIIIb is a GPI-linked receptor lacking a cytoplasmic tail and with no other subunits known to associate with it, its signaling mechanism is only partially described. Recently, it was reported that the signal pathway, activated by FcgRIIIb leading to NET formation, involves Syk, TAK1 (transforming growth factor-b-activated kinase 1), the MEK (ERK kinase)/ERK (extracellular signal-regulated kinase) cascade, activation of PKC, and activation of the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase complex, which produce reactive oxygen species (ROS) (18). Therefore, it is possible that FcgRIIIb uses some of these signaling molecules to induce an increase in [Ca 2+ ] i . Transient Receptor Potential Melastatin 2 (TRPM2), a nonselective Ca 2+ -permeable membrane cation channel, is highly expressed in myeloid cells (19,20). This receptor is a member of the TRP family of cation-selective channels that are weakly voltage-sensitive and diversely opened by temperature, mechanical force, pH, and oxidative stress (21,22). TRPM2 can be opened through directly binding with intracellular adenosine diphosphate ribose (ADPR) (23) and can also be indirectly activated under conditions of oxidative stress (24,25), acidification (26), and elevated intracellular Ca 2+ (27). Additionally, in dorsal root ganglion neurons, the activity of TRPM2 is increased by the addition of phorbol 12-myristate 13acetate (PMA) which leads to activation of PKC (28,29). Thus, because TRPM2 can be activated by ROS and PKC, two of the second messengers involved in the signaling pathway from FcgRIIIb leading to NET formation (18), it is possible that TRPM2 is used by FcgRIIIb to induce an increase in [Ca 2+ ] i . In order to test this hypothesis, both FcgRIIa and FcgRIIIb were stimulated by specific monoclonal antibodies, and the increase in [Ca 2+ ] i was measured in the presence or absence of pharmacological inhibitors. The neutrophil chemoattractant fMLF (N-formyl-methionil-leucyl-phenylalanine), and also FcgRIIa induced a rapid increase in [Ca 2+ ] i . FcgRIIIb aggregation also induced an increase in [Ca 2+ ] i , but this increase was delayed by several seconds. Despite both, FcgRIIa and FcgRIIIb aggregation-induced ROS production, in the presence of diphenyleneiodonium chloride (DPI), an inhibitor of the NADPH-oxidase complex (30), only the FcgRIIIb-induced increase in [Ca 2+ ] i was reduced. Similarly, in the presence of Gö6976, an inhibitor of PKC (31,32), only FcgRIIIb-induced increase in [Ca 2+ ] i was reduced. In addition, 2-amino ethoxydiphenyl borate (2-APB) (33)(34)(35) and clotrimazole (36)(37)(38)(39), inhibitors of TRPM2, reduced FcgRIIIb-induced, but not FcgRIIa-induced increase in [Ca 2+ ] i . These data show for the first time that FcgRIIIb aggregation activates TRPM2 via PKC and ROS for inducing an increase in [Ca 2+ ] i in human neutrophils.

Fluorescent Calcium Measurements
Increase of cytosolic calcium concentration was measured by detecting fluorescence changes in neutrophils loaded with Fura 2-AM as previously described (45,46). Briefly, neutrophils were loaded with 10 mM Fura-2, washed, and resuspended (1.5 x 10 6 cell/ml) in PBS with 1.5 mM Ca 2+ and 1.5 mM Mg 2+ and kept on ice. Then, fluorescent changes of a 2-ml stirred neutrophil suspension kept at 37°C were monitored with a LS55 spectrofluorimeter (Perking Elmer; Waltham, MA, USA), using 340 and 380 nm excitation wavelengths and 510 nm emission wavelength. Calcium concentrations were calculated as described by Grynkiewicz et al. (47), using the software FL WinLab 4.00.02 (Perking Elmer; Waltham, MA, USA).
For fMLF stimulation, 3 x 10 6 Fura-2-loaded neutrophils in 2 ml of PBS with Ca 2+ and Mg 2+ were placed in a spectrofluorimeter cuvette and incubated at 37°C for 2 min. After that, fluorescence changes were recorded for 100 s, then 40 ml of 500 nM fMLF were added (final concentration of 10 nM). For FcgR stimulation, 3 x 10 6 Fura-2-loaded neutrophils were first resuspended in 100 ml PBS containing 10 mg/ml of the corresponding anti-FcgR antibody, and incubated on ice for 20 min. After one wash in cold PBS, neutrophils were resuspended in 2 ml PBS with Ca 2+ and Mg 2+ and transferred to a spectrofluorimeter cuvette. Fluorescence changes were recorded for 100 s, and then 80 ml of 1.3 mg/ml F(ab') 2 goat anti-mouse IgG (final concentration 52 mg/ml) were added to aggregate the receptors.

Measurement of Reactive Oxygen Species (ROS)
ROS production was assessed by detecting fluoresce changes in neutrophils loaded with dihydrorhodamine 123 (DHR-123). Neutrophils (1 x 10 6 ) were resuspended in 100 ml of 15 mM DHR-123 in PBS and incubated for 15 min at 37°C in the dark. Neutrophils were washed with 1 ml PBS, and then resuspended in 100 ml of PBS with the corresponding stimulus as follows. For PMA, neutrophils were resuspended in PBS containing 20 nM of PMA, and then incubated at 37°C in the dark for 45 min. For FcgR stimulation, neutrophils were resuspended in PBS containing 10 mg/ml of the corresponding anti-FcgR antibody, and incubated on ice for 20 min. Next, neutrophils were washed in cold PBS, resuspended in 100 ml of PBS containing 10 mg/ml F (ab') 2 goat anti-mouse IgG, and incubated at 37°C in the dark for 45 min. After incubation, 250 ml cold PBS were added and cells kept on ice for 2 min. Finally, neutrophils were centrifuged and resuspended in 1% paraformaldehyde in PBS at 4°C. Neutrophils were stored cold in the dark until analyzed by flow cytometry using an Attune NxT Flow Cytometer (Thermo Fisher Scientific), with the 485 nm (excitation) and 520 nm (emission) filters. Neutrophils were gated by dot-plot analysis and 10,000 cells were acquired per sample. Data analysis was performed using the Flowjo X software (Tree Star Inc., Ashland, OR, USA).

Statistical Analysis
Quantitative data were expressed as mean ± standard error of mean (SEM). Single variable data were compared by pairedsample Student's t-tests using the computer program KaleidaGraph ® version 4.5.2 for Mac (Synergy Software; Reading, PA, USA). Also, multiple pair-comparisons were performed using Tukey's test after ordinary one-way analysis of variance (ANOVA). Post hoc differences were considered statistically different at a value p < 0.05.

Fcg Receptors Induce an Increase in Cytoplasmic Ca 2+ Concentration
Neutrophils, the most abundant leukocytes in blood, display important functions for controlling infections, inflammation, and immune responses (2,48). Several of these functions are initiated by antibodies binding to Fc receptors (6). Although, both Fcg receptors on human neutrophils (FcgRIIa and FcgRIIIb) are known to activate particular cell responses, the signaling capabilities of each receptor are still incompletely understood (9). About 30 years ago, it was already known that both neutrophil   (15). Despite these early studies the mechanism used by FcgRIIIb to mobilize calcium is still unknown. Thus, in order to further explore the mechanisms of [Ca 2+ ] i increase induced by Fcg receptors, Fura-2-loaded neutrophils were first stimulated with the chemoattractant fMLF. In response to fMLF a rapid almost immediate increase in [Ca 2+ ] i was observed ( Figure 1A). After this rise, the [Ca 2+ ] i then returned to basal levels ( Figure 1A). Similarly, aggregation of the FcgRIIa with specific monoclonal antibodies also induced a rapid increase in [Ca 2+ ] i ( Figure 1B). Also, aggregation of the FcgRIIIb with specific monoclonal antibodies resulted in an increase in [Ca 2+ ] i ( Figure 1C). Although, the magnitude of the [Ca 2+ ] i rise was similar with all three stimuli ( Figure 1D), the FcgRIIIb-mediated increase in [Ca 2+ ] i was delayed for about 10 to 20 seconds ( Figure 1C), suggesting again that the mechanism for calcium mobilization was different between the two Fcg receptors.

FcgRIIIb Aggregation Induces an Increase in [Ca 2+ ] i via Ca 2+ Entry From Extracellular Medium
It is well established that fMLF-induced increase in [Ca 2+ ] i has two components, one initial release of Ca 2+ from internal stores followed by a subsequent influx of this cation from outside the cell by the mechanism known as store-operated calcium entry (SOCE) (49,50). Similarly, it is believed that Fcg receptors also display a similar mechanism for an increase in [Ca 2+ ] i (51,52). Thus, we sought to confirm these ideas by selectively stimulating each Fcg receptor. Human neutrophils were placed in Ca 2+ -free medium and then stimulated with fMLF. As expected an initial increase in [Ca 2+ ]i was observed followed by a gradual return to basal levels around 90 seconds later ( Figure 1E). At this time and excess of Ca 2+ was added to the medium. This led to a second increase in [Ca 2+ ] i , which represents influx of Ca 2+ from outside the cell ( Figure 1E). Similarly, in Ca 2+ -free medium aggregation of FcgRIIa induced an increase in [Ca 2+ ] i from internal stores that returned to basal levels after about 90 seconds ( Figure 1F). When Ca 2+ was restored to the medium a second increase in [Ca 2+ ] i , was also observed ( Figure 1F), although this second rise was much smaller. This indicated that similarly to the fMLF, aggregation of FcgRIIa induced a release of Ca 2+ from internal stores and then an influx of Ca 2+ from outside the cell. In sharp contrast, in Ca 2+ -free medium aggregation of FcgRIIIb did not induce any increase in [Ca 2+ ] i ( Figure 1G), suggesting that no Ca 2+ was released from intracellular stores. When Ca 2+ was restored to the medium an important increase in [Ca 2+ ] i , was detected ( Figure 1G). After fMLF stimulation, the magnitude of the [Ca 2+ ] i rise from internal stores was very similar to the increase in [Ca 2+ ] i from outside the cell ( Figure 1H). Each part was about half of the total increase in calcium observed in cells kept in Ca 2+ -containing medium. For FcgRIIa stimulation, the magnitude of the [Ca 2+ ] i rise from internal stores was similar to the increase in [Ca 2+ ] i induced by fMLF ( Figure 1H). The [Ca 2+ ] i rise from extracellular Ca 2+ was smaller than the [Ca 2+ ] i rise from internal stores ( Figure 1H), suggesting that the major contribution to an increase in [Ca 2+ ] i rise after FcgRIIa engagement was from internal stores. In contrast, the FcgRIIIb-mediated increase in [Ca 2+ ] i was almost exclusively due to influx of extracellular Ca 2+ ( Figure 1H). These data suggested that contrary to other Fcg receptors, FcgRIIIb induces only an influx of Ca 2+ from outside the cell.  (Figure 2A) nor FcgRIIIb aggregation ( Figure 2B). Similarly, UO126, a selective MEK inhibitor did not affect the increase in [Ca 2+ ] i induced by either Fcg receptor ( Figure 2). Additionally, as expected neither LLZ 1640-2 nor UO126 affected the increase in [Ca 2+ ] i induced by fMLF stimulation ( Figure 2C). However, since the fMLF receptor is a GPCR (53) treatment with Pertussis toxin completely blocked the increase in [Ca 2+ ] i ( Figures 2C, D).
In contrast, Pertussis toxin did not affect the increase in [Ca 2+ ] i induced by either Fcg receptor ( Figure 2D). In the past, we have reported that FcgRIIIb signaling involves Syk, PKC, and NADPH-oxidase (12,18). Therefore, we explored the possible involvement of these signaling molecules in FcgRIIIb-mediated increase in [Ca 2+ ] i . Treatment with iSyk, a selective Syk inhibitor, reduced the increase in [Ca 2+ ] i induced by either FcgRIIa (Figure 3A) or FcgRIIIb aggregation ( Figure 3B). Additionally, Gö6976 a selective PKC inhibitor blocked the increase in [Ca 2+ ] i induced by FcgRIIIb but not by FcgRIIa (Figure 3). Similarly, DPI a selective NADPH-oxidase inhibitor reduced the increase in [Ca 2+ ] i induced by FcgRIIIb but not by FcgRIIa ( Figure 3). These data showed that Syk was involved in both FcgRIIa-and FcgRIIIb-mediated increase in [Ca 2+ ] i , and suggested for the first time that FcgRIIIb signaling, but not FcgRIIa signaling to increase [Ca 2+ ] i in human neutrophils involves both PKC and NADPH-oxidase.

FcgRIIIb-Induced ROS Production Involves Syk and PKC
It has also been shown that NAPDH-oxidase is an enzymatic complex responsible for ROS production in human neutrophils  (54,55), and PKC is able to induce NAPDH-oxidase activation (56,57). Because, FcgRIIIb signaling involves both PKC and NADPH-oxidase activation (12,18), we explored whether FcgRIIIb requires PKC to induce ROS production. Neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA), a strong PKC activator, produced a robust amount of ROS (Figure 4). Similarly, activation of FcgRIIa or FcgRIIIb resulted in significant ROS production, although smaller than the one induced by PMA   Figure 4). When cells were pre-treated with iSyk, a selective Syk inhibitor, PMA-induced ROS production was not affected (Figure 4). In contrast, iSyk reduced ROS production induced by either FcgRIIa or FcgRIIIb ( Figure 4). Also, in the presence of Gö6976, a selective PKC inhibitor, PMA-induced ROS production was blocked (Figure 4). Similarly, Gö6976 completely prevented ROS production after aggregation of FcgRIIa or FcgRIIIb ( Figure 4). These data suggested that FcgRIIa and FcgRIIIb both activate Syk, leading to PKC activation and ROS production. However, only in FcgRIIIb signaling these molecules are connected to a rise in [Ca 2+ ] i ( Figure 3C).   (58). In neutrophils several TRP channels were found to be expressed at the mRNA level by RT-PCR, including TRPC6, TRPM2, TRPV1, TRPV2, TRPV5 and TRPV6 (59). Of these channels, TRPM2 was demonstrated to   Figure 5). In contrast, 2-APB efficiently inhibited the FcgRIIIb-mediated increase in [Ca 2+ ] i ( Figure 5).
Similarly, the FcgRIIa-mediated [Ca 2+ ] i rise was not affected by the presence of clotrimazole ( Figure 5). However, clotrimazole also efficiently inhibited the FcgRIIIb-mediated increase in [Ca 2+ ] i ( Figure 5). Moreover, 2-APB could not inhibit ROS production induced by PMA ( Figure 6) nor by FcgRIIa or  FcgRIIIb ( Figure 6). These data positioned production of ROS up-stream of TRPM2 activation and suggested that PKC and ROS are required for FcgRIIIb-induced activation of TRPM2 to promote Ca 2+ influx in neutrophils (Figure 7).

DISCUSSION
Neutrophils represent the most abundant leukocytes in blood and are considered the first line of defense against invading microorganisms because they are the first leukocytes to arrive at sites of inflammation and infection (63)(64)(65). At affected sites neutrophils display several antimicrobial functions (66) including degranulation, production of reactive oxygen species (ROS) (67,68), phagocytosis (4) and the formation of neutrophil extracellular traps (NET) (5). In addition to these innate immune functions, neutrophils also participate in modulating the adaptive immune response (2). Initiation of these multiple cellular functions involves numerous receptors triggering a myriad of intracellular signaling pathways (52). In many of these pathways, changes in intracellular Ca 2+ concentration ([Ca 2+ ] i ) are a prerequisite for neutrophil activation (52,69). However, how changes in [Ca 2+ ] i induced by the multitude of receptors expressed on these cells, control neutrophil activation and function remains only partially understood (58,70,71). In the case of antibody-mediated neutrophil responses (8,9) (52,69), and consequently Ca 2+ fluxes for neutrophil responses are finely regulated in terms of temporal and spatial organization (58). In resting conditions, [Ca 2+ ] i in neutrophils is around 100 nM, a level 10 000-fold lower than the concentration in the extracellular medium (74). Upon stimulation of neutrophils via various receptors such as Gprotein coupled receptors (GPCRs) (75), integrins (76), or Fcg receptors (45,72) there is a rapid increase in [Ca 2+ ] i caused by the release of Ca 2+ from intracellular stores and/or by influx of extracellular Ca 2+ . Engagement of these receptors leads to activation of phospholipase C (PLC). GPCRs mainly activate the PLCb2 and PLCb3 (75), while integrins and Fcg receptors  activate PLCg1 and PLCg2 (52,76). PLC in turn act on phosphatidylinositol 4,5 bisphosphate (PIP 2 ) to generate diacylglycerol (DAG) and IP 3 . Binding of IP 3 to its cognate receptor (IP 3 R), which also functions as a nonselective Ca 2+ channel, localized on the membrane of the endoplasmic reticulum (ER), leads to a release of Ca 2+ into the cytoplasm (41,77). The initial rapid release of Ca 2+ from the ER is followed by influx of Ca 2+ across the plasma membrane. This influx is induced by the drop in Ca 2+ levels inside the ER in a process that is known as store-operated calcium entry (SOCE) (78,79). The mechanism for SOCE involves [Ca 2+ ] i sensing proteins such as stromal interaction molecule 1 (STIM1), which migrates from the ER to the plasma membrane when intracellular stores are discharged (78). At the plasma membrane STIM1 gets together with the Ca 2+ channel protein Orai1 (71,80), which allows influx of extracellular Ca 2+ . In addition, it has been observed that after receptor stimulation there is also a Ca 2+ influx that is dependent on receptor occupation by agonists and relatively store independent. This other mechanism of Ca 2+ entry is known as receptor-operated calcium entry (ROCE) (81,82). The molecular mechanisms controlling these two components of Ca 2+ influx are still not resolved (83,84).
Although the main mechanism for increasing [Ca 2+ ] i in neutrophils is primarily mediated via IP 3 -dependent Ca 2+ release from intracellular stores followed by SOCE activation of Orai1 channels (16), there is evidence that additional ion channels via ROCE are also involved in calcium influx into these cells (58). This has been clearly demonstrated for fMLF stimulation. After the initial increase in [Ca 2+ ] i a second influx of extracellular Ca 2 + is observed. This influx is composed predominantly by Orai1 channels which are selective for Ca 2+ , and also by other nonselective channels that allow entry of both Ca 2+ and strontium cations (Sr 2+ ) (50). In phagocytes, the nature of Ca 2+ channels mediating ROCE is just beginning to be identified.
An interesting group of nonselective ion channels that may participate in ROCE mechanisms in phagocytes is the superfamily of transient receptor potential (TRP) channels (85). TRP channels are expressed in many cell types and participate in multitude of physiological and pathological processes, such as cell proliferation, differentiation, and death (21). They are particularly important as biosensors of environmental and cellular stimuli including heat, mechanical force (pressure), oxidative (redox) status, and pH (21,22,86). The family of TRP channels is divided in six subfamilies: the ankyrin (TRPA), the canonical (TRPC), the melastatin (TRPM), the mucolipin (TRPML), the polycystin (TRPP) and the vanilloid (TRPV) subfamilies. Human and murine neutrophils only express members of the TRPC, TRPM and TRPV subfamilies (58), but their role in ROCE is unclear (81). In mast cells, a ROCE mechanism was described after antigen stimulation. The channel involved allowed influx of both external Ca 2+ and Sr 2+ to support degranulation, and was identified as TRPC5 (canonical transient receptor potential channel 5) (87). These reports opened the possibility that TRP channels may contribute to changes in [Ca 2+ ] i after Fcg receptor engagement in human neutrophils.
In phagocytic cells, it is clear that changes in [Ca 2+ ] i are important for antibody-mediated cell responses such as phagocytosis (45,72) and ROS production (67). It is generally accepted that Fcg receptors activate a signaling cascade that involves Src family kinase-mediated phosphorylation of an ITAM sequence (7) in the cytoplasmic portion of the receptor (or its associated g chains) (6,8). The phosphorylated ITAM becomes a docking site for Syk, which in turn activates PLCg1 to produce IP 3 (17,52). This pathway is certainly the one used by FcgRIIa to induce Ca 2+ release from the ER (15). Then, a SOCE mechanism composed by STIM1 and Orai1 proteins is activated to generate a further influx of Ca 2+ from outside the cell (16,71). This influx of extracellular Ca 2+ was found to be important for the intraphagosomal production of ROS during phagocytosis of opsonized yeast particles (51). We indeed found that selective aggregation of FcgRIIa produces an increase in [Ca 2+ ] i resulting from release of Ca 2+ from intracellular stores (Figure 1). For FcgRIIa it is clear that increases in [Ca 2+ ] i involve IP 3 -mediated Ca 2+ release from intracellular stores, followed by a SOCE mechanism that allows Ca 2+ entry from outside the cell (71,88).
For FcgRIIIb, elucidating the mechanism for initiating an increase in [Ca 2+ ] i has been more complicated, since this receptor is expressed exclusively on human neutrophils, and it is a glycosylphosphatidylinositol (GPI)-linked receptor, lacking transmembrane and cytoplasmic domains (8,9). Despite that initial steps in signaling for this receptor remain a mystery, it is clear that it can trigger signaling pathways leading to different neutrophil responses including activation of integrins (89), activation of transcription factors (11), and induction of NET formation (12,13). In addition, FcgRIIIb induces an increase in [Ca 2+ ] i (14,45,90), which is independent of IP 3 (15). For a long time, it has been assumed that the initial rise in [Ca 2+ ] i must be due to release of Ca 2+ from intracellular stores by another mechanism that is independent of IP 3 . However, no such mechanism has yet been found. Sphingosine 1 phosphate (S1P), the product of sphingosine kinase (SK) is considered to be a mediator for changes in [Ca 2+ ] i . In neutrophils, S1P formation was dependent on ER store depletion, and inhibition of SK resulted in a reduction of Ca 2+ influx (91). Also, in glioblastoma cells it was reported that S1P could activate the TRP channel TRPC1, leading to Ca 2+ influx (92). In both cases, S1P was found to mediate Ca 2+ entry into the cells and not release of Ca 2+ from intracellular stores. Therefore, it is unlikely that S1P mediates release of Ca 2+ from intracellular stores in neutrophils. Now, we report that when neutrophils were in Ca 2+ -free medium selective aggregation of FcgRIIIb did not cause any increase in [Ca 2+ ] i (Figure 1). This result implied that the increase in [Ca 2+ ] i was due to an influx of Ca 2+ from outside the cell. This idea was confirmed when Ca 2+ was restored in to the medium (Figure 1). This finding is in complete agreement with the lack of IP 3 production when FcgRIIIb is engaged on neutrophils (15). It also pointed to the idea that a membrane ion channel was activated in response to FcgRIIIb aggregation. In previous reports, we described that FcgRIIIb triggers a signaling cascade that involves Syk, TAK1, MEK-ERK for induction of NETosis (18). In addition, we also reported that activation of PKC and production of ROS are important for NET formation (12). Thus, we explored whether these molecules could be involved in FcgRIIIb-mediated increase in [Ca 2+ ] i . Indeed, we found that PKC and ROS are required for the influx of Ca 2+ induced by FcgRIIIb (Figure 3).
Based on this, we turned our attention to TRP channels which are non-selective ion channels allowing transport of Ca 2+ (85,93), and capable (some of them) to sense the redox status in the cell (86,94). Human neutrophils express the TRP channels: TRPC1, TRPC3, TRPC4, TRPC6, TRPM2, TRPV1, TRPV2, TRPV4, TRPV5, and TRPV6 (58,59). Among these receptors, only TRPM2 (transient receptor potential melastatin type 2 cation channel; previously also named as TRPC7 or LRPC2) (20,58) is also known to be activated by PKC (28,29), and ROS (24,95). In addition, TRPM2 has been found to be involved in several immune functions, including clearance of bacteria (96)(97)(98), NET formation by murine neutrophils (99), activation of NLRP3 inflammasome and secretion of interleukin-1b (100,101), and dendritic cell maturation and chemotaxis (102). We confirmed that TRPM2 is responsible for FcgRIIIb-mediated rise in [Ca 2+ ] i when two inhibitors, 2-APB (33)(34)(35)95) and clotrimazole (36)(37)(38)(39) completely blocked FcgRIIIb-mediated rise in [Ca 2+ ] i ( Figure 5). Both inhibitors 2-APB and clotrimazole have been used to block TRPM2 in many cell systems, however they are not specific inhibitors of this channel. Therefore, the possibility remains that other TRP channels may be involved in FcgRIIIb-mediated Ca 2+ influx. Recently, novel and potentially more specific TRPM2 inhibitors have been reported (103,104). It would be interesting to use these novel inhibitors to confirm our conclusions. However, we feel confident that TRPM2 is in fact the channel involved because it is the only TRP channel member on human neutrophils that is activated by PKC and ROS. In fact, the mechanism of activation involves adenosine diphosphate ribose (ADPR) binding to the Cterminal domain of the receptor, which presents strong homology to the human nucleotide diphosphate linked moiety X type (Nudix) hydrolase motif 9 (NUDT9) (23,105). The NADPH-oxidase, like the mitochondrial oxidase, is a molecular complexes vectorially arranged on a membrane such that it accepts an electron from NADPH in the cytosol and transfers it across the membrane reducing oxygen to an oxygen radical (55,68). The NADPH-oxidase is usually assembled on the phagosomal membrane, to generate ROS into the phagosome, or on the plasma membrane to generate extracellular ROS. Since, oxidative stress induces intracellular accumulation of ADPR it would also be interesting to confirm that aggregation of FcgRIIIb indeed causes an accumulation of ADPR. It is also important to establish whether ADPR is indeed coming from activation of the NADPH-oxidase or from mitochondria in response to oxidative stress. TRPM2 gating requires in addition to ADPR, binding of Ca 2+ (61,106,107). This requirement for full opening of the channel has been very nicely revealed through analysis of cryo-electron microscopy structures of human and zebrafish TRPM2 [reviewed in (108,109)]. In the case, of FcgRIIIb-mediated TRPM2 activation, we do not know if the basal [Ca 2+ ] i is sufficient to support the full opening of the channel. In future experiments, we will eliminate intracellular calcium with BAPTA to further explore the mechanism for Ca 2+ entry in neutrophils after FcgRIIIb engagement. Also, we will look at the functional consequence of the calcium rise on particular FcRIIIb-mediated neutrophil responses.
The involvement of PKC for inducing activation of TRPM2 comes from indirect studies using PMA (28,29). In this report, we also demonstrated that indeed PKC is involved in FcgRIIIbmediated TRPM2 activation using the specific PKC inhibitor Gö6976 ( Figure 3). However, the particular isoform of PKC that is required for this function remains undetermined. Human neutrophils expressed PKC isoforms from each group of PKC enzymes (110). The inhibitor Gö6976 has specificity for Ca 2+dependent PKC isozymes alpha and beta (31). The most likely PKC isoform involved in this response may be PKCb, since this isoform is an upstream mediator of NADPH-oxidase activation and was reported to be involved in NET formation (111); and we have also reported that FcgRIIIb is the main Fc receptor involved in NETosis (12). Future experiments will determine whether PKCb is actually required for FcgRIIIb-mediated TRPM2 activation. Tremendous advances have taken place in the field of Ca 2+ signaling in neutrophils in recent years. However, the literature in this area of research is rather controversial, as human and murine neutrophils and even human HL-60 cells do not always express the same ion channels on their membrane (58,88). Thus, there is difficulty in integrating the different findings between species, and between cell lines and primary cells. Still, this field has a bright future since there are still many open questions on how calcium signals regulate neutrophil functions (70).
In conclusion, we have found that FcgRIIIb does not induce Ca 2+ release from intracellular stores but it does activate, via PKC and ROS, the TRPM2 channel on the plasma membrane for inducing an influx of extracellular Ca 2+ into human neutrophils (Figure 7).

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Bioethics Committee at Instituto de Investigaciones Biomedicas -Universidad Nacional Autónoma de Mexico (UNAM). The participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
OA performed most of the experiments and analyzed data. NM performed calcium measurements. CR designed the research, mentored other authors, performed statistical analysis, prepared figures, organized the references, and wrote the paper. All authors contributed to the article and approved the submitted version.