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Differential regulation of human neutrophil FcgammaRIIa (CD32) and FcgammaRIIIb (CD16)-induced Ca2+ transients.

Human neutrophils express two structurally distinct receptors for the Fc region of IgG, FcgammaRIIa and FcgammaRIIIb. Although earlier studies have suggested that the functional properties of these receptors are similar, mounting evidence suggests that these receptors are capable of inducing distinct functional responses. Accordingly, we have examined the regulation of intracellular Ca2+ transients induced by each of these receptors alone (homotypic receptor cross-linking) and together (heterotypic receptor cross-linking). The glycosylphosphatidylinositol-anchored FcgammaRIIIb induces a rise in [Ca2+] after homotypic cross-linking that is independent of ligand-mediated engagement of the transmembrane FcgammaRIIa. Both receptors were sensitive to the protein-tyrosine kinase inhibitor methyl 2,5-dihydroxycinnamate, but FcgammaRIIa-induced signaling was uniquely sensitive to the protein-tyrosine kinase inhibitor genistein. FcgammaRIIa but not FcgammaRIIIb engages a cAMP-sensitive and inositol 1,4, 5-trisphosphate-dependent pathway(s) that results in the Ca2+-transient. When these receptors are cross-linked into heterotypic clusters, a synergistic rise in [Ca2+] is observed that is accompanied by synergistic increases in the phospholipase Cgamma-breakdown products inositol 1,4,5-trisphosphate and diglyceride. These data provide a mechanism for the functional differences between these two receptors and suggest the possibility that they can be differentially modulated.

Receptors for the Fc region of IgG (Fc␥R) 1 are critical participants in inflammation and in the immune response by providing an important link between the humoral and cellular immune systems. The cluster of eight genes for human Fc␥R on chromosome 1q encode a diverse group of receptors that display similar extracellular domains yet remarkably diverse transmembrane and cytoplasmic domains (1)(2)(3). Human neutrophils constitutively express two distinct Fc␥R: Fc␥RIIa and Fc␥RIIIb. Fc␥RIIa is a transmembrane receptor that can initiate many neutrophil inflammatory responses including degranulation and the generation of reactive oxygen intermediates. Fc␥RIIIb is a glycosylphosphatidylinositol (GPI)-linked protein that can also initiate a number of neutrophil inflammatory responses.
Tyrosine phosphorylation events are essential for the early intracellular signals initiated by Fc␥R. Fc␥RIIa has an immunoreceptor tyrosine activation motif in the cytoplasmic domain (4), and mutational analysis has shown the importance of the tyrosine residues in this motif for the functional capacity of this receptor (5)(6)(7). Cross-linking of Fc␥RIIa results in the association of the receptor with src-family tyrosine kinases (fgr in PMN, lyn and hck in THP-1 cells) and Syk (p72 syk ) (8 -10). In myeloid cell lines, Fc␥RIIa-induced activation of PLC␥ by Syk results in a rapid IP 3 -mediated [Ca 2ϩ ] transient (11,12). The mechanisms for early tyrosine phosphorylation events triggered after cross-linking of Fc␥RIIIb are less clear. Most likely the result of preferential partitioning of GPI-anchored proteins and palmitylated src-family kinases in lipid domains in the plasma membrane (13), Fc␥RIIIb, like many GPI-anchored proteins (14), is associated with an src-family kinase hck in certain detergent-insoluble complexes (15).
An early view that Fc␥RIIIb is simply a binding molecule without signaling capacity (16,17) has been revised by ample evidence that Fc␥RIIIb activates protein-tyrosine kinases and initiates intracellular [Ca 2ϩ ] i transients, degranulation, and the respiratory burst (15, 18 -20). Although some Fc␥RIIIb functions may overlap with Fc␥RIIa, Fc␥RIIIb does have a distinct repertoire of cell programs that it initiates. Unlike Fc␥RIIa, Fc␥RIIIb does induce a unique proinflammatory phenotype in neutrophils (21). Although it is not a phagocytic receptor (17,22), Fc␥RIIIb enhances Fc␥RIIa-mediated internalization and functions cooperatively with CD11b/CD18 in promoting phagocytosis and the respiratory burst (22)(23)(24). Therefore, using changes in the intracellular [Ca 2ϩ ] i levels, which are induced by Fc␥RIIa and Fc␥RIIIb and which are required for many receptor functions (5,25,26), we have explored the possibility of differential regulation of signaling by Fc␥RIIa and Fc␥RIIIb. Both receptors elicit a brisk increase in [Ca 2ϩ ] i derived primarily from intracellular stores. Unlike Fc␥RIIa, which engages a cAMP-sensitive IP 3 -dependent pathway for generation of [Ca 2ϩ ] i transients, Fc␥RIIIb engages a cAMP-insensitive pathway that is also resistant to the proteintyrosine kinase inhibitor genistein. These two distinct pathways can interact synergistically at the level of phosphatidylinositol 4, 5-bisphosphate breakdown to lead to enhanced transients in [Ca 2ϩ ] i , reflecting both intracellular and extracellular stores. Engagement of these two distinct pathways may provide the basis for different cell programs initiated by these receptors.

EXPERIMENTAL PROCEDURES
Reagents and Buffers-All buffers and solutions were made with ultra-purified endotoxin-free water (Millipore). Glassware was rendered endotoxin-free by either washing in chromic acid/nitric acid or by baking at 190°C for 4 h. A modified PBS solution was prepared with 5 mM KCl and 5 mM glucose. Modified PBS plus Ca 2ϩ and Mg 2ϩ included 1.0 mM CaCl 2 and 1.65 mM MgCl 2 . Solutions were confirmed to have Ͻ0.05 endotoxin units/ml by the limulus lysate assay (Associates of Cape Cod). Indo-1 acetoxymethyl ester (Molecular Probes, Eugene, OR), a cell permeant fluorogenic Ca 2ϩ indicator, was prepared as a 0.5 mM stock in absolute ethanol. mAb IV.3, a murine IgG2b recognizing human Fc␥RII (CD32), was obtained as both purified IgG and purified Fab fragments (Medarex, Annendale, NJ). mAb 3G8, a murine IgG1 recognizing human Fc␥RIII, was obtained as purified F(abЈ) 2 fragments or purified IgG (Medarex). mAb 41H16 IgG, a murine IgG2a that preferentially recognizes the R131 allele of human Fc␥RIIA (27), was kindly provided by Dr. Thomas Zipf (University of Texas Cancer Center, Houston, TX). Goat F(abЈ) 2 fragments specific for murine IgG gamma and light chains (GAM) (TAGO Immunologicals, Burlingame, CA and Jackson ImmunoResearch, West Grove, PA) were obtained in both unconjugated and phycoerythrin-conjugated forms. Ab Fab/F(abЈ) 2 fragments contained no detectable intact IgG or heavy chains as judged by silver stain SDSpolyacrylamide gel electrophoresis and by size exclusion high performance liquid chromatography analysis.
Preparation of PMN-Fresh heparinized blood from healthy donors was diluted with an equal volume of modified PBS at 25°C, and PMN were separated from the diluted blood by a two-step discontinuous density gradient consisting of ficoll-hypaque (density ϭ 1.075 and 1.125 g/ml) (28). After two washes with modified PBS, the cells were treated with distilled water for 15 s to lyse contaminating erythrocytes, followed by an equal volume of 1.8% saline solution to restore isotonicity. The remaining PMN were resuspended in modified PBS at 1 ϫ 10 7 cells/ml. By microscopic examination Ͼ95% of the cells were PMN. Separations were completed within 2 h, and all experimental procedures were completed within 5-6 h of phlebotomy.
Donors were typed for the Fc␥RIIa-H131/R131 polymorphism by a combination of mAb reactivity (using mAbs 41H16 and IV.3 exactly as described (27)) and/or by DNA genotyping using allele-specific PCR reactions (29). There is complete concordance between these two assays.
Analysis of Intracellular Ca 2ϩ Concentrations-Indo-1, a fluorescent dye with spectral properties that change with the binding of free Ca 2ϩ , was used to measure changes in intracellular calcium concentrations as we have described (5,18). PMN were incubated at 37°C for 15 min with 5 M indo-1 AM. After loading, the cells were washed once with modified PBS and maintained at 25°C in the dark. In most experiments, an aliquot of cells (at a concentration of 1 ϫ 10 7 cells/ml) was opsonized with anti-Fc␥R mAb for 5 min at 37°C followed by one wash at room temperature. The cells were then resuspended to 5 ϫ 10 6 cells/ml in modified PBS, and an aliquot was removed to quantitate mAb opsonization levels by indirect immunofluorescence (see below). The cells were then warmed to 37°C for 5 min in modified PBS plus 1.1 mM Ca 2ϩ and 1.6 mM Mg 2ϩ before analysis. Cells were loaded in an identical manner with fura-2 AM (2 M) for single-cell Ca 2ϩ analysis (see below).
Indo-1 fluorescence analysis was performed on an SLM 8000C Spectrofluorometer (SLM-Aminco, Urbana, IL). Excitation at 355 nm was provided by a xenon arc lamp and a monochromator, whereas emission at 405 and 490 nm were simultaneously monitored with two monochromators and photomultiplier tubes. A corresponding stimulus was injected into each cuvette at 60 s without interruption of acquisition. Constant temperature (37°C) and stirring was maintained throughout each experiment. Each sample was individually calibrated for both maximal and minimum indo-1 fluorescence by the sequential addition of Triton X-100 and EDTA, and the 405/490 nm ratio was converted to [Ca 2ϩ ] as described previously (5,18).
For analysis of intracellular Ca 2ϩ transients induced by the opsonized E, 100 l of fura-2-loaded PMN (1.5 ϫ 10 6 cell/ml) were added to a 25-mm-diameter round glass coverslip and allowed to settle for 15 min at 37°C. During the last 5 min, 1.1 mM Ca 2ϩ and 1.6 mM Mg 2ϩ was added. The coverslips were then transferred to the stage of a Nikon Diaphot (Nikon), and the ratio of fluorescence emission of fura-2 was monitored. After the establishment of a base line, E-IV.3 or E-3G8 F(abЈ) 2 were added. Analysis was continued for an additional 5 min.
Quantitation of IP 3 and Diglyceride Formation-To quantitate stimulus-induced changes in [IP 3 ], isolated PMN (pre-equilibrated with Ca 2ϩ /Mg 2ϩ as described above) were mixed with mAb 3G8 IgG, fMLP, or opsonized E (see above) for various periods of time followed by rapid addition of ice-cold 15% trichloroacetic acid (TCA). Alternatively, cells were pre-opsonized with anti-Fc␥R mAb Fab or F(abЈ) 2 fragments for 5 min at 37°C. After one wash, cells were re-equilibrated with Ca 2ϩ /Mg 2ϩ and then stimulated with F(abЈ) 2 GAM and incubated for various periods of time followed by the rapid addition of ice-cold 15% trichloroacetic acid. In both cases, the precipitates were pelleted in a microfuge for 15 min at 4°C. The supernatants were extracted three times with 10 volumes of water-saturated diethyl ether and neutralized to pH 7.5 with NaHCO 3 . IP 3 levels were quantitated by competitive receptor binding assay with [ 3 H]IP 3 and IP 3 -binding protein (30) exactly according to the manufacturer's instructions (Amersham Pharmacia Biotech).
Diglyceride mass in total lipid extracts was determined by conversion to [ 32 P]phosphatidic acid with [ 32 P]orthophosphate (Amersham) and diglyceride kinase (Calbiochem) following the method of Preiss et al. (31). Briefly, cells were prepared and stimulated exactly as described above for quantitation of IP 3 levels except that 5 g/ml cytochalsin B was included during the final 5-min equilibration with Ca 2ϩ /Mg 2ϩ . After various periods of time, cells were rapidly lysed with 50 volumes of iced 2:1 methanol:chloroform. Extracts were processed as described previously (32).
Preparation of mAb Opsonized E-Biotinylated mAb IV.3 Fab, mAb 3G8 F(abЈ) 2 , and bovine erythrocytes (E) were prepared as we have previously described (33). Biotinylated E were saturated with streptavidin and washed. The resulting E were coated with biotinylated mAb, and the level of mAb binding was verified by flow cytometry. For E-IV. 3 Fab or E-3G8 F(abЈ) 2 -induced stimulation of IP 3 and diglyceride production, E were added to PMN suspensions at a ratio of 25:1 (E:PMN) and gently pelleted for 15 s followed by incubation at 37°C for various periods of time.
Immunofluorescent Flow Cytometry-Aliquots of PMN at 5 ϫ 10 6 cell/ml were incubated with saturating concentrations of primary mAb for 30 min at 4°C. After two washes, the cells were incubated with saturating concentrations of phycoerythrin-conjugated goat anti-mouse IgG F(abЈ) 2 at 4°C for another 30 min. In addition, cells obtained from each [Ca 2ϩ ] i measurement test cuvette were directly stained with saturating amounts of phycoerythrin-conjugated goat anti-mouse IgG F(abЈ) 2 at 4°C for 30 min. After washing, the cells were analyzed immediately for immunofluorescence using a Cytofluorograf IIS flow cytometer and a 2151 computer (Becton Dickinson Immunocytometry Systems, Westwood, MA).

Homotypic Fc␥RIIa and Fc␥RIIIb Ca 2ϩ
Transients-Fc␥Rmediated neutrophil stimulation activates the respiratory burst, which is dependent on receptor-induced elevations in the intracellular [Ca 2ϩ ]. Since Fc␥RIIa and Fc␥RIIIb associate with different tyrosine kinases (15), we hypothesized that the rise in intracellular Ca 2ϩ induced by these structurally distinct receptors might be differentially regulated. Accordingly, using anti-receptor mAb Fab and F(abЈ) 2 fragments, we developed an experimental system to cross-link these receptors in a receptorspecific manner involving one type (homotypic) or both types (heterotypic) of receptors. When either neutrophil Fc␥RIIa or Fc␥RIIIb are cross-linked with anti-receptor mAb Fab or F(abЈ) 2 fragments (homotypic cross-linking), a brisk rise in [Ca 2ϩ ] is observed (Fig. 1A). Indeed, the rise in [Ca 2ϩ ] is similar in magnitude to the flux observed in response to the potent neutrophil-activating peptide fMLP (Fig. 1A). This rise in [Ca 2ϩ ] is due to release of Ca 2ϩ from intracellular stores. When either EDTA or EGTA is added to the extracellular media, the Fc␥R and fMLP-mediated [Ca 2ϩ ] fluxes are intact (results not shown) (18,34). The quantitative level of the Fc␥R-induced Ca 2ϩ flux is dependent on the concentration of the stimulating anti-receptor mAb. Over a subsaturating range of mAb concentrations, a dose response in the quantitative rise in [Ca 2ϩ ] was observed (Fig. 1B), and at saturating concentrations of antireceptor mAb, the rise in [Ca 2ϩ ] induced by cross-linking Fc␥RIIIb is consistently higher in peak magnitude than the rise induced by cross-linking Fc␥RIIa (1417 Ϯ 183 versus 846 Ϯ 82 nM peak rise in [Ca 2ϩ ], Fc␥RIIa versus Fc␥RIIIb respectively, p Ͻ 0.005, n ϭ 20).
The Fc␥RIIa-induced rise in [Ca 2ϩ ] is dependent on the integrity of the immunoreceptor tyrosine activation motif in the cytoplasmic domain (5-7). The mechanism by which the GPI-anchored Fc␥RIIIb induces functional responses (such as the rise in [Ca 2ϩ ]) is less clear. One possible explanation for the mAb 3G8 F(abЈ) 2 ϩ F(abЈ) 2 GAM-induced rise in [Ca 2ϩ ] is through ligand-dependent engagement of Fc␥RIIa. This could occur if either the anti-Fc␥RIIIb mAb F(abЈ) 2 or the crosslinking GAM F(abЈ) 2 contained intact IgG molecules. Although SDS-polyacrylamide gel electrophoresis analysis did not indicate the presence of any intact IgG under nonreducing conditions or of undigested heavy chain under reducing conditions in our Fab or F(abЈ) 2 preparations, we prepared biotinylated anti-Fc␥RIIa Fab and Fc␥RIIIb F(abЈ) 2 fragments, and when neutrophils were opsonized with either biotinylated mAb, a rise in [Ca 2ϩ ] was observed upon addition of streptavidin ( Fig. 2A). These data exclude the possibility that the homotypic Fc␥RIIIb-induced rise in Ca 2ϩ is the result of contaminating IgG in the F(abЈ) 2 GAM preparation.
To exclude the possibility that the mAb 3G8 F(abЈ) 2 preparation contained residual IgG that could engage Fc␥RIIa, PMN were first saturated with unlabeled mAb IV.3 Fab. To confirm that any IgG remaining in our 3G8 F(abЈ) 2 preparation could be blocked from engaging Fc␥RIIa by IV.3 Fab, we first examined the mAb 3G8 IgG-induced rise in Ca 2ϩ . This mAb induces formation of heterotypic Fc␥RIIa ϩ Fc␥RIIIb clusters (5), and as expected, the magnitude of this response is sensitive to the Fc␥RIIa-H131/R131 polymorphism; the IgG1 mAb 3G8 binds Fc␥RIIa-H131 poorly and induces a diminished Ca 2ϩ transient relative to that observed in Fc␥RIIa-R131/R131 donors (Fig.  2B). Preincubation with mAb IV.3 Fab completely blocks this mAb 3G8 IgG-induced Ca 2ϩ transient, demonstrating that heterotypic cross-linking of both receptors is required for this response (Fig. 2B). mAb 3G8 F(abЈ) 2 alone (resulting in monoor bivalent engagement of Fc␥RIIIb) does not elicit a Ca 2ϩ transient (Fig. 2B). When neutrophils were preincubated mAb IV.3 Fab (to block the ligand binding site of Fc␥RIIa), the streptavidin-induced 3G8 F(abЈ) 2 -biotin Ca 2ϩ transient was unaltered (Fig. 2C). Additional controls included the complete blockade of the biotinylated IV.3-Fab ϩ streptavidin-induced Ca 2ϩ transient by preincubation with IV.3 Fab. Also, preincubation of PMN with unlabeled mAb 3G8 F(abЈ) 2 did not alter the biotinylated IV.3-Fab ϩ streptavidin-induced rise in [Ca 2ϩ ] but did block the mAb 3G8 F(abЈ) 2 -biotin-induced [Ca 2ϩ ] transient (Fig. 2C). These results categorically demonstrate that homotypic cross-linking of Fc␥RIIIb results in a rise in [Ca 2ϩ ] that is independent of ligand-mediated interactions with the transmembrane Fc␥RIIa.
Differential Regulation of the Fc␥RIIa and Fc␥RIIIb Ca 2ϩ Transients-Cross-linking of neutrophil Fc␥R results in tyrosine kinase activity (3,15,22). The dependence of the Fc␥RIIIbinduced Ca 2ϩ transient on protein-tyrosine kinase activity was shown with the tyrosine kinase inhibitor methyl 2,5-dihydroxycinnamate (100 M), a stable erbstatin analog; the Fc␥RIIaand Fc␥RIIIb-mediated rise in [Ca 2ϩ ] was completely blocked by pretreatment with this protein-tyrosine kinase inhibitor (Fig. 3). Cell viability was unaltered by the brief exposure (5 min) to this tyrosine kinase inhibitor as determined by exclusion of trypan blue (control, 90% cells viable; treated cells, 85% saturation ϭ 0.5 g/ml (100%) and mAb 3G8 F(abЈ) 2 binding saturation ϭ 1 g/ml (100%)). A representative experiment of four is shown. Comparable results were obtained with the tyrosine kinase inhibitors tyrophostin (40 g/ml, n ϭ 3), lavendustin A (50 g/ml, n ϭ 2), 2-hydroxy-5-(2,5-dihydroxybenzyl)aminobenzoic acid (1 g/ml, n ϭ 3), and staurosporine, which inhibits both tyrosine and ser/thr kinases (0.5 g/ml, n ϭ 3). However, differential sensitivity to the tyrosine kinase inhibitor genistein was observed for the Fc␥RII-and Fc␥RIII-mediated [Ca 2ϩ ] transients. When neutrophils were incubated with 100 M genistein for 5 min, the rise in [Ca 2ϩ ] induced by cross-linking Fc␥RIIa (Fig. 3) and by fMLP (results not shown) was completely abolished. Surprisingly, the Fc␥RIIIb-induced rise in [Ca 2ϩ ] was not abrogated by 100 M genistein (Fig. 3). In four independent paired experiments, the ability of Fc␥RIIa but not Fc␥RIIIb to initiate a rise in [Ca 2ϩ ] was abrogated by 100 M genistein (Fc␥RIIa/Fc␥RIIIb % control, 5 Ϯ 4%/52 Ϯ 6%; n ϭ 4, p Ͻ 0.001). The differential sensitivity to genistein was also observed at 50 M genistein (Fc␥RIIa/Fc␥RIIIb % control, 26 Ϯ 3%/63 Ϯ 2%, n ϭ 3, p Ͻ 0.001). These concentra- Alternatively, PMN from a donor homozygous for Fc␥RIIa-R131/R131 were directly stimulated with mAb 3G8 F(abЈ) 2 . Finally, the 3G8 IgG-induced Ca 2ϩ transient in PMN from an Fc␥RIIa-R131/R131 homozygous was completely blocked by preincubation of the cells with mAb IV.3 Fab (2 g/ml). A representative experiment of six is shown. C, cells were preincubated with the indicated nonbiotinylated mAb, then opsonized with mAb IV.3 Fab-biotin or mAb 3G8 F(abЈ) 2 -biotin. After one wash to remove unbound mAb, the blocking nonbiotinylated mAb was re-added (to ensure compete blockade throughout the entire experiment), and the cells were stimulated at 60 s with streptavidin. A representative experiment of three is shown. tions of genistein have been shown to completely inhibit neutrophil Fc␥RIIa-induced tyrosine phosphorylation and phagocytosis (37,38) and to block neutrophil degranulation and superoxide production in response to cross-linking of Fc␣R and L-selectin (35,36). Little or no sensitivity of the Fc␥RIIa-or Fc␥RIIIb-induced Ca 2ϩ transient to the tyrosine kinase inhibitor reduced carboxamidomethylated and maleylated-lysozyme (100 g/ml, n ϭ 2) or the ser/thr kinase inhibitors calphostin C, H-7, H-8, and H-1004 was observed. These results demonstrate that both Fc␥RIIa and Fc␥RIIIb initiate Ca 2ϩ transients in a tyrosine kinase-dependent manner. Despite this similarity, the differential sensitivity to the tyrosine kinase inhibitor genistein suggests that these receptors are engaging distinct intracellular activation pathways.
It has been shown that neutrophil Fc␥R-induced superoxide production and phagocytosis are inhibited by occupancy of the adenosine A 2 receptor (39 -41). Because these responses are dependent on a rise in [Ca 2ϩ ], we tested the susceptibility of Fc␥R-specific [Ca 2ϩ ] transients to the potent adenosine A 2 receptor agonist NECA. Neutrophils were treated with varying concentrations of NECA for 5 min at 37°C before Fc␥R crosslinking. In the presence of 10 Ϫ6 M NECA (the same concentration required for inhibition of Fc␥R phagocytosis (39)), the rise in [Ca 2ϩ ] induced by cross-linking of Fc␥RIIa was significantly inhibited (38.0 Ϯ 4.3% of control, n ϭ 4, p Ͻ 0.01) (Fig. 4A). In contrast, the Fc␥RIIIb-mediated rise in [Ca 2ϩ ] was unaltered by the same concentrations of NECA (98.1 Ϯ 21.5% of control, n ϭ 4, p Ͼ 0.05), demonstrating differential regulation of Fc␥RII and Fc␥RIII signaling by this adenosine A 2 agonist (Fc␥RIIa versus Fc␥RIIIb, n ϭ 4, p Ͻ 0.01).
Engagement of adenosine A 2 receptors has been shown to lead to transient increases in intracellular levels of cAMP, increases in intracellular [Ca 2ϩ ] in mast cells, and to activation of a membrane-associated serine/threonine protein phosphatase (42)(43)(44). The ability of NECA to activate PLC␥ and increase [Ca 2ϩ ] may be the basis for the inhibition of the Fc␥RIIa-induced Ca 2ϩ transient (cf. Fig. 4A). However, in human neutrophils, the magnitude of the NECA-induced Ca 2ϩ transient is only a fraction of the level of the Fc␥RIIa-induced transient (40 Ϯ 6 nM rise in Ca 2ϩ after stimulation with 10 Ϫ6 M NECA, n ϭ 3). Alternatively, it is known that inhibition of Fc␥R phagocytosis by adenosine A 2 receptors is mediated at least in part by increases in intracellular levels of cAMP (40).

IP 3 -dependent and -independent Ca 2ϩ Transients-Elevations in [cAMP]
can induce activation of the cAMP-dependent protein kinase, which in turn can down-modulate PLC␥1 activity (46). The sensitivity of the Fc␥RIIa-induced, but not the Fc␥RIIIb-induced, rise in [Ca 2ϩ ] to cAMP suggests that Fc␥RIIa may engage an IP 3 -dependent mechanism. Upon maximal homotypic Fc␥RIIIb cross-linking with saturating levels of mAb 3G8 F(abЈ) 2 and F(abЈ) 2 GAM, there was no detectable increase in IP 3 levels, which is in marked distinction to the time-dependent increase in IP 3 observed after homotypic crosslinking of Fc␥RIIa with mAb Fab and F(abЈ) 2 GAM (Table I). As a positive control, the fMLP-induced increase in IP 3 levels is shown (Table I).
We considered the possibility that the effectiveness of receptor cross-linking might be an important variable. Accordingly, we prepared erythrocytes opsonized with saturating levels of mAb IV.3 Fab or mAb 3G8 F(abЈ) 2 using a biotin-avidin coupling technique. The ability of these probes (E-IV. 3 Fab and E-3G8 F(abЈ) 2 for Fc␥RII and Fc␥RIII, respectively) to crosslink their respective receptors and elicit a rise in [Ca 2ϩ ] was confirmed by single cell analysis of fura-2-loaded neutrophils (Fig. 5). However, using the mAb-coated E as a stimulus, engagement of Fc␥RIIa but not Fc␥RIIIb induced an increase in [IP 3 ] (results not shown).
Biochemical Characterization of the Heterotypic Fc␥RIIϩ-Fc␥RIII Ca 2ϩ Transient-Our results with receptor-specific IP 3 data do not provide an explanation for the vigorous IP 3 response that has been reported during antibody opsonized erythrocyte (EA) phagocytosis (47). EA can engage both Fc␥RIIa and Fc␥RIIIb, resulting in heterotypic cross-linking of these receptors. Since heterotypic cross-linking of Fc␥RIIa and Fc␥RIIIb results in a synergistic phagocytic response (22) 3. Differential sensitivity of the Fc␥RIIa-and Fc␥RIIIbinduced Ca 2؉ transient to inhibition by the protein-tyrosine kinase inhibitor genistein. Cells were opsonized with the indicated anti-Fc␥R mAb as described under "Experimental Procedures." After one wash, cells were resuspended in buffer containing Ca 2ϩ /Mg 2ϩ and genistein, or methyl 2,5-dihydroxycinnamate was added. After a 5-minute incubation at 37°C, data acquisition was initiated. F(abЈ) 2 GAM was added as stimulus at 60 s. A representative experiment of three is shown.

FIG. 4. Differential sensitivity of the Fc␥RIIa-and Fc␥RIIIb-induced Ca 2؉ transient to agents that increase intracellular [cAMP].
A, cells were prepared as described in Fig. 3 except that 10 Ϫ6 M NECA was included during the final 5-min preincubation before data acquisition. A representative experiment of four is shown. B, cells were preincubated for 30 min at 37°C with Bt 2 cAMP followed by mAb opsonization as described in Fig. 1. A representative experiment of five is shown. C, cells were prepared as described in Fig. 3 except that 10 Ϫ6 M MP and 10 Ϫ4 M isobutylmethylxanthine was included during the final 5-min preincubation before data acquisition. A representative experiment of six is shown. IBMX, isobutylmethylxanthine. ergistic rise in [Ca 2ϩ ] (Fig. 6).
To determine if the heterotypic Fc␥R cross-linking results in an IP 3 response that is distinct from that elicited by homotypic receptor cross-linking, IP 3 responses were quantitated after heterotypic cross-linking of Fc␥RIIa and Fc␥RIIIb by 3G8 IgG (to avoid pre-opsonization, which can significantly increase base-line IP 3 levels (Table I)). In marked contrast to homotypic cross-linking of either receptor, heterotypic Fc␥R cross-linking (in a Fc␥RIIa-R131/R131 donor) resulted in a significantly enhanced IP 3 . In fact, the time-dependent increase in IP 3 is very similar to the fMLP response both temporally and in magnitude and more than 2-fold higher than the maximal homotypic Fc␥RIIa response (Fig. 7A, Table I). In parallel with the synergistic IP 3 production, significantly elevated levels of diglycerides were detected after heterotypic receptor cross-linking in a time manner that is similar in magnitude to the fMLP response (Fig. 7B). Resting diglyceride levels were the range of 30 -40pmol/10 6 cells, in agreement with the range reported in the literature (48), and increased 2-3-fold upon stimulation with heterotypic Fc␥R cross-linking or fMLP. A comparable increase in diacylglyceride levels was also detected in mAb 3G8 IgG-stimulated cells (21.4 Ϯ 4.3, 58.2 Ϯ 3.9, 73.2 Ϯ 2.1, 41.2 Ϯ 11.4 pmol/10 6 cells at t ϭ 0, 1, 5, and 10 min, respectively (n ϭ 3)). Homotypic receptor cross-linking did not result in any detectable increase in diglyceride levels. These data show that heterotypic Fc␥R cross-linking results in a synergistic Ca 2ϩ

FIG. 5. Single cell analysis of E-3G8 F(ab) 2 (A) or E-IV.3 Fab (B) stimulated PMN.
PMN were placed on a 25-mm-diameter coverslip and allowed to settle for 15 min at 37°C as described under "Experimental Procedures." The cells were placed on the microscope stage, and a field was defined in which there were more than 20 cells. Data acquisition was started, and after base-line determination for 10 s, mAb-opsonized E were added. Each line represents an individual cell. The heterogeneous response is due to the asynchronous binding of the opsonized E to the PMN.
FIG. 6. Fc␥RIIa and Fc␥RIIIb heterotypic cross-linking induces a synergistic Ca 2؉ response. Cells were opsonized with the indicated levels of mAb (B) and the GAM-induced Ca 2ϩ response was measured (A). To achieve identical mAb opsonization densities, mAb IV.3 Fab was used at saturation (0.5 g/ml) for Fc␥RIIa homotypic cross-linking, mAb 3G8 F(abЈ) 2 was used at a subsaturating dose (0.3 g/ml) for Fc␥RIIIb homotypic cross-linking, and the heterotypic cross-linking was induced by using 0.25 g/ml mAb IV.3 Fab and 0.15 g/ml mAb 3G8 F(abЈ) 2 . A representative experiment of five is shown.

TABLE I Quantification of PMN IP 3 levels after homotypic
Fc␥R cross-linking or fMLP stimulation Neutrophils were prepared and stimulated with fMLP or the indicated mAb (IV.3 Fab, 0.5 g/ml; 3G8 F(abЈ) 2 , 2 g/ml; and F(abЈ) 2 GAM, 35 g/ml) for varying periods of time. Cells were rapidly pelleted, and IP 3 levels were determined in cell extracts using a competitive IP 3 receptor binding assay as described under "Experimental Procedures." Values represent mean Ϯ S.D. (n ϭ 5).  3 ]. These data demonstrate that the biochemical regulation of Fc␥RIIIb function is distinct from Fc␥RIIa, and they provide the initial basis for understanding the distinct repertoire of cell programs initiated by Fc␥RIIIb.
Fc␥RIIa interacts with the src-family tyrosine kinase fgr and with Syk through interactions between the phosphorylated immunoreceptor tyrosine activation motif in the cytoplasmic domain of Fc␥RIIa and SH2 domains in the kinases (3,12). One characteristic of Syk activation is the tyrosine phosphorylation and activation of PLC␥, resulting in the breakdown of phosphatidylinositol 4,5-bisphosphate into IP 3 and diacylglyceride (49,50). Indeed, in myelomonocytic cell lines, cross-linking of Fc␥RIIa is associated with a rapid rise in [IP 3 ] (11,12). Data suggesting that stimulation of Fc␥R in human neutrophils does not lead to any change in the intracellular [IP 3 ] (34, 51), a finding at variance with our results (Table I, Fig. 7), may reflect technical differences in the threshold for detection. We chose to use an indirect receptor binding assay to quantitate IP 3 levels, which avoids the biosynthetic labeling of cells with myo-[ 3 H]inositol, a process that is inherently inefficient and difficult to perform in neutrophils due to the necessarily short labeling periods.
Heterotypic neutrophil Fc␥R cross-linking during Fc␥R-mediated phagocytosis or immune complex-induced Fc␥R-stimulation, elicits an IP 3 burst (47,52). Our data indicate that the nature of the Fc␥R stimulus is critical; there is no detectable increase in IP 3 levels after homotypic cross-linking of Fc␥RIIIb, a small but detectable increase in [IP 3 ] after cross-linking of Fc␥RIIa, and a substantial generation of IP 3 induced by heterotypic cross-linking of neutrophil Fc␥R with the IP 3 response, comparable in magnitude to the fMLP-induced IP 3 response. In parallel with the increased [IP 3 ], we also observed significant increases in the concentration of diacylglyceride, the other breakdown product of phosphatidylinositol 4,5-bisphosphate.
These data provide a mechanism for the synergism between Fc␥RIIa and Fc␥RIIIb in the generation of the early Ca 2ϩ transient ( Fig. 6) (53) and perhaps for phagocytosis (22) and the oxidative burst (23). Since cross-linking of Fc␥RIIIb results in tyrosine phosphorylation of Fc␥RIIa (22), enhanced tyrosine phosphorylation of Fc␥RIIa might enhance the ability of this receptor to activate downstream effector molecules such as PLC␥, leading to the IP 3 and diacylglyceride responses observed after heterotypic receptor cross-linking.
The ability of GPI-anchored proteins to generate intracellular signals is now well established (14). Although the mechanisms may not be completely understood, current data suggest that these proteins are capable of activating src-family kinases. Some evidence suggests that GPI-anchored proteins and myristylated src-family kinases are both found in specialized lipid domains in the plasma membrane. Indeed, neutrophil Fc␥RIIIb co-precipitates in detergent-insoluble domains with hck, which is in contrast to the association of neutrophil Fc␥RIIa with fgr (15). Differential association and activation of src-kinases by neutrophil Fc␥RIIa and Fc␥RIIIb may provide an explanation for the differences in sensitivity to the protein-tyrosine kinase inhibitor genistein. These data also demonstrate that it may be possible to differentially manipulate the functional capacity of these receptors, a property that may be useful in altering the response of neutrophils to circulating IgG autoantibodies such an anti-neutrophil cytoplasmic antibodies. Selective inactivation of Fc␥RIIIb, which plays an important role in anti-neutrophil cytoplasmic antibodies-positive Wegener's granulomatosis (21,54), might allow targeted down-modulation of neutrophilmediated injury mechanisms in that disease.
Although our results clearly show that Fc␥RIIa does induce IP 3 production, Fc␥RIIIb does not elicit an IP 3 response after homotypic cross-linking (Table I). This lack of detectable IP 3 cannot be due to a lack of sensitivity, since homotypic engagement of Fc␥RIIIb at receptor saturation consistently results in a Ca 2ϩ transient that is higher in magnitude than the response elicited by homotypic cross-linking of Fc␥RIIa. Nonetheless, the Fc␥RIIIb-induced Ca 2ϩ is released from intracellular stores (18). The nature of the intracellular Ca 2ϩ -mobilizing signal has yet to be elucidated. Among the IP 3 -independent mechanisms, cyclic ADP-ribose and sphingosine-1-phosphate are candidates for intracellular Ca 2ϩ -mobilizing signals (55)(56)(57). Of course, it is also possible that Fc␥RIIa engages both IP 3 -dependent and IP 3independent Ca 2ϩ -releasing mechanisms. Future studies will be needed to resolve the role of IP 3 , cyclic ADP-ribose, and sphingosine kinase in neutrophil Fc␥R-mediated Ca 2ϩ transients.
There are significant implications in the finding that Fc␥RIIIb does not engage an IP 3 -mediated signaling pathway. Direct interactions between GPI-anchored proteins and srcfamily kinases provide one possible mechanism for the transmission of intracellular signal generation. Alternatively, GPIanchored proteins may interact with transmembrane proteins to form multimolecular complexes. The formation of multimolecular complexes in the membrane is a common theme among plasma membrane receptors, including Fc␥RIa, Fc␥RIIIa, Fc␣RI, and Fc⑀RI (1, 3). The nature and identity of possible Fc␥RIIIb-associating structures is currently unclear. Elegant co-capping and fluorescence resonance energy transfer studies have shown that CD11b/CD18 in neutrophil membranes can associate with a wide range of other cell surface receptors including Fc␥RIIIb, leukocyte function antigen-1 (LFA-1), and the urokinase receptor (58 -60). However, the lack of an Fc␥RIIIb-induced increase in IP 3 contrasts to the ability of CD11b/CD18 to induce increases in [IP 3 ] after cross-linking (47). Furthermore, the ability of Fc␥RIIIb to activate CD11b/ CD18 for phagocytosis, a function that CD11b/CD18 cannot do alone in resting neutrophils, indicates that all Fc␥RIIIb signaling cannot be mediated through CD11b/CD18 (22).
Our results also provide the basis for understanding that the results of Fc␥R engagement on neutrophils will depend on which receptor type(s) are engaged. An IgG2 ligand will selectively and homotypically engage Fc␥RIIa of the H131 genotype (61,62). Anti-neutrophil cytoplasmic antibodies may favor engagement of the more highly expressed Fc␥RIIIb. In contrast, multivalent immune complexes would favor heterotypic crosslinking of Fc␥RIIa, Fc␥RIIIb, and perhaps complement receptors as well. Each of these might result in the engagement of different biochemical signal-transducing pathways and in qualitatively and quantitatively different effector functions. Delineation of receptor-specific pathways is essential in the identification of kinases and kinase substrates that are important in regulation of Fc␥R-mediated signal transduction. Ultimately, an understanding of these pathways will enable specific modulation of Fc␥R-initiated inflammatory processes in autoimmune diseases without complete blockade of all Fc␥R-mediated functions, which may be essential in normal host defense.