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J Biol Chem, Vol. 273, Issue 14, 8071-8079, April 3, 1998

Differential Regulation of Human Neutrophil Fcgamma RIIa (CD32) and Fcgamma RIIIb (CD16)-induced Ca2+ Transients*

Jeffrey C. EdbergDagger , James J. Moon§, David J. Chang, and Robert P. Kimberly

From the Division of Clinical Immunology and Rheumatology, Departments of Medicine and Microbiology, University of Alabama, Birmingham, Alabama 35294

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Human neutrophils express two structurally distinct receptors for the Fc region of IgG, Fcgamma RIIa and Fcgamma RIIIb. 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 Fcgamma RIIIb induces a rise in [Ca2+] after homotypic cross-linking that is independent of ligand-mediated engagement of the transmembrane Fcgamma RIIa. Both receptors were sensitive to the protein-tyrosine kinase inhibitor methyl 2,5-dihydroxycinnamate, but Fcgamma RIIa-induced signaling was uniquely sensitive to the protein-tyrosine kinase inhibitor genistein. Fcgamma RIIa but not Fcgamma RIIIb 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.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Receptors for the Fc region of IgG (Fcgamma 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 Fcgamma R on chromosome 1q encode a diverse group of receptors that display similar extracellular domains yet remarkably diverse transmembrane and cytoplasmic domains (1-3). Human neutrophils constitutively express two distinct Fcgamma R: Fcgamma RIIa and Fcgamma RIIIb. Fcgamma RIIa is a transmembrane receptor that can initiate many neutrophil inflammatory responses including degranulation and the generation of reactive oxygen intermediates. Fcgamma 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 Fcgamma R. Fcgamma 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 (5-7). Cross-linking of Fcgamma 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 (p72syk) (8-10). In myeloid cell lines, Fcgamma RIIa-induced activation of PLCgamma by Syk results in a rapid IP3-mediated [Ca2+] transient (11, 12). The mechanisms for early tyrosine phosphorylation events triggered after cross-linking of Fcgamma 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), Fcgamma 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 Fcgamma RIIIb is simply a binding molecule without signaling capacity (16, 17) has been revised by ample evidence that Fcgamma RIIIb activates protein-tyrosine kinases and initiates intracellular [Ca2+]i transients, degranulation, and the respiratory burst (15, 18-20). Although some Fcgamma RIIIb functions may overlap with Fcgamma RIIa, Fcgamma RIIIb does have a distinct repertoire of cell programs that it initiates. Unlike Fcgamma RIIa, Fcgamma RIIIb does induce a unique proinflammatory phenotype in neutrophils (21). Although it is not a phagocytic receptor (17, 22), Fcgamma RIIIb enhances Fcgamma RIIa-mediated internalization and functions cooperatively with CD11b/CD18 in promoting phagocytosis and the respiratory burst (22-24). Therefore, using changes in the intracellular [Ca2+]i levels, which are induced by Fcgamma RIIa and Fcgamma RIIIb and which are required for many receptor functions (5, 25, 26), we have explored the possibility of differential regulation of signaling by Fcgamma RIIa and Fcgamma RIIIb. Both receptors elicit a brisk increase in [Ca2+]i derived primarily from intracellular stores. Unlike Fcgamma RIIa, which engages a cAMP-sensitive IP3-dependent pathway for generation of [Ca2+]i transients, Fcgamma RIIIb engages a cAMP-insensitive pathway that is also resistant to the protein-tyrosine 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 [Ca2+]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
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 Ca2+ and Mg2+ included 1.0 mM CaCl2 and 1.65 mM MgCl2. Solutions were confirmed to have <0.05 endotoxin units/ml by the limulus lysate assay (Associates of Cape Cod). Indo-1 acetoxymethyl ester (Molecular Probes, Eugene, OR), a cell permeant fluorogenic Ca2+ indicator, was prepared as a 0.5 mM stock in absolute ethanol.

mAb IV.3, a murine IgG2b recognizing human Fcgamma RII (CD32), was obtained as both purified IgG and purified Fab fragments (Medarex, Annendale, NJ). mAb 3G8, a murine IgG1 recognizing human Fcgamma RIII, was obtained as purified F(ab')2 fragments or purified IgG (Medarex). mAb 41H16 IgG, a murine IgG2a that preferentially recognizes the R131 allele of human Fcgamma RIIA (27), was kindly provided by Dr. Thomas Zipf (University of Texas Cancer Center, Houston, TX). Goat F(ab')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(ab')2 fragments contained no detectable intact IgG or heavy chains as judged by silver stain SDS-polyacrylamide gel electrophoresis and by size exclusion high performance liquid chromatography analysis.

The protein-tyrosine kinase inhibitors (genistein, methyl 2,5-dihydroxycinnamate, tyrophostin, lavendustin A, 2-hydroxy-5-(2,5-dihydroxybenzyl)aminobenzoic acid and staurosporine) were obtained from Life Technologies, Inc.. All other kinase and phosphatase inhibitors were from Calbiochem. Misoprostol (MP) was the kind gift of Dr. Barbara Struthers (G. D. Searle). Remaining reagents were from Sigma.

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 = 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 × 107 cells/ml. By microscopic examination >95% 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 Fcgamma 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 Ca2+ Concentrations-- Indo-1, a fluorescent dye with spectral properties that change with the binding of free Ca2+, 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 µM 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 × 107 cells/ml) was opsonized with anti-Fcgamma R mAb for 5 min at 37 °C followed by one wash at room temperature. The cells were then resuspended to 5 × 106 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 Ca2+ and 1.6 mM Mg2+ before analysis. Cells were loaded in an identical manner with fura-2 AM (2 µM) for single-cell Ca2+ 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 [Ca2+] as described previously (5, 18).

For analysis of intracellular Ca2+ transients induced by the opsonized E, 100 µl of fura-2-loaded PMN (1.5 × 106cell/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 Ca2+ and 1.6 mM Mg2+ 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(ab')2 were added. Analysis was continued for an additional 5 min.

Quantitation of IP3 and Diglyceride Formation-- To quantitate stimulus-induced changes in [IP3], isolated PMN (pre-equilibrated with Ca2+/Mg2+ 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-Fcgamma R mAb Fab or F(ab')2 fragments for 5 min at 37 °C. After one wash, cells were re-equilibrated with Ca2+/Mg2+ and then stimulated with F(ab')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 NaHCO3. IP3 levels were quantitated by competitive receptor binding assay with [3H]IP3 and IP3-binding protein (30) exactly according to the manufacturer's instructions (Amersham Pharmacia Biotech).

Diglyceride mass in total lipid extracts was determined by conversion to [32P]phosphatidic acid with [32P]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 IP3 levels except that 5 µg/ml cytochalsin B was included during the final 5-min equilibration with Ca2+/Mg2+. 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(ab')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(ab')2-induced stimulation of IP3 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 × 106 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(ab')2 at 4 °C for another 30 min. In addition, cells obtained from each [Ca2+]i measurement test cuvette were directly stained with saturating amounts of phycoerythrin-conjugated goat anti-mouse IgG F(ab')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).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Homotypic Fcgamma RIIa and Fcgamma RIIIb Ca2+ Transients-- Fcgamma R-mediated neutrophil stimulation activates the respiratory burst, which is dependent on receptor-induced elevations in the intracellular [Ca2+]. Since Fcgamma RIIa and Fcgamma RIIIb associate with different tyrosine kinases (15), we hypothesized that the rise in intracellular Ca2+ induced by these structurally distinct receptors might be differentially regulated. Accordingly, using anti-receptor mAb Fab and F(ab')2 fragments, we developed an experimental system to cross-link these receptors in a receptor-specific manner involving one type (homotypic) or both types (heterotypic) of receptors. When either neutrophil Fcgamma RIIa or Fcgamma RIIIb are cross-linked with anti-receptor mAb Fab or F(ab')2 fragments (homotypic cross-linking), a brisk rise in [Ca2+] is observed (Fig. 1A). Indeed, the rise in [Ca2+] is similar in magnitude to the flux observed in response to the potent neutrophil-activating peptide fMLP (Fig. 1A). This rise in [Ca2+] is due to release of Ca2+ from intracellular stores. When either EDTA or EGTA is added to the extracellular media, the Fcgamma R and fMLP-mediated [Ca2+] fluxes are intact (results not shown) (18, 34). The quantitative level of the Fcgamma R-induced Ca2+ 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 [Ca2+] was observed (Fig. 1B), and at saturating concentrations of anti-receptor mAb, the rise in [Ca2+] induced by cross-linking Fcgamma RIIIb is consistently higher in peak magnitude than the rise induced by cross-linking Fcgamma RIIa (1417 ± 183 versus 846 ± 82 nM peak rise in [Ca2+], Fcgamma RIIa versus Fcgamma RIIIb respectively, p < 0.005, n = 20).


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  		Fig. 1.   Ca2+ transients in human neutrophils induced by fMLP and Fcgamma R cross-linking. A, in the upper tracing, cells were stimulated with 10-7 M fMLP. In the middle and lower tracings, cells were opsonized with 2 µg/ml anti-Fcgamma RIII mAb 3G8 F(ab')2 or 1 µg/ml anti-Fcgamma RII mAb IV.3 Fab, respectively, washed to remove unbound mAb, and then stimulated with F(ab')2 GAM (35 µg/ml) at 60 s. A representative experiment of 20 is shown. B, the relationship between peak [Ca2+] and anti-receptor mAb IV.3 Fab or mAb 3G8 F(ab')2 concentration. The peak Ca2+ level was determined as described in panel A. The concentration of mAb relative to the saturating concentration (determined by flow cytometric analysis) is shown (mAb IV.3 Fab binding saturation = 0.5 µg/ml (100%) and mAb 3G8 F(ab')2 binding saturation = 1 µg/ml (100%)). A representative experiment of four is shown.

The Fcgamma RIIa-induced rise in [Ca2+] is dependent on the integrity of the immunoreceptor tyrosine activation motif in the cytoplasmic domain (5-7). The mechanism by which the GPI-anchored Fcgamma RIIIb induces functional responses (such as the rise in [Ca2+]) is less clear. One possible explanation for the mAb 3G8 F(ab')2 + F(ab')2 GAM-induced rise in [Ca2+] is through ligand-dependent engagement of Fcgamma RIIa. This could occur if either the anti-Fcgamma RIIIb mAb F(ab')2 or the cross-linking GAM F(ab')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(ab')2 preparations, we prepared biotinylated anti-Fcgamma RIIa Fab and Fcgamma RIIIb F(ab')2 fragments, and when neutrophils were opsonized with either biotinylated mAb, a rise in [Ca2+] was observed upon addition of streptavidin (Fig. 2A). These data exclude the possibility that the homotypic Fcgamma RIIIb-induced rise in Ca2+ is the result of contaminating IgG in the F(ab')2 GAM preparation.


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  		Fig. 2.   Homotypic Fcgamma R cross-linking induces a rise in intracellular [Ca2+]. A, cells were opsonized with the indicated biotinylated anti-receptor mAb, washed, and stimulated by the addition of streptavidin at 60 s. A representative experiment of four is shown. B, PMN from a Fcgamma RIIa-R131/R131 and a Fcgamma RIIa-H131/H131 homozygous donors were isolated and directly stimulated with mAb 3G8 IgG in the upper two tracings. Alternatively, PMN from a donor homozygous for Fcgamma RIIa-R131/R131 were directly stimulated with mAb 3G8 F(ab')2. Finally, the 3G8 IgG-induced Ca2+ transient in PMN from an Fcgamma RIIa-R131/R131 homozygous was completely blocked by preincubation of the cells with mAb IV.3 Fab (2 µg/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(ab')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.

To exclude the possibility that the mAb 3G8 F(ab')2 preparation contained residual IgG that could engage Fcgamma RIIa, PMN were first saturated with unlabeled mAb IV.3 Fab. To confirm that any IgG remaining in our 3G8 F(ab')2 preparation could be blocked from engaging Fcgamma RIIa by IV.3 Fab, we first examined the mAb 3G8 IgG-induced rise in Ca2+. This mAb induces formation of heterotypic Fcgamma RIIa + Fcgamma RIIIb clusters (5), and as expected, the magnitude of this response is sensitive to the Fcgamma RIIa-H131/R131 polymorphism; the IgG1 mAb 3G8 binds Fcgamma RIIa-H131 poorly and induces a diminished Ca2+ transient relative to that observed in Fcgamma RIIa-R131/R131 donors (Fig. 2B). Preincubation with mAb IV.3 Fab completely blocks this mAb 3G8 IgG-induced Ca2+ transient, demonstrating that heterotypic cross-linking of both receptors is required for this response (Fig. 2B). mAb 3G8 F(ab')2 alone (resulting in mono- or bivalent engagement of Fcgamma RIIIb) does not elicit a Ca2+ transient (Fig. 2B). When neutrophils were preincubated mAb IV.3 Fab (to block the ligand binding site of Fcgamma RIIa), the streptavidin-induced 3G8 F(ab')2-biotin Ca2+ transient was unaltered (Fig. 2C). Additional controls included the complete blockade of the biotinylated IV.3-Fab + streptavidin-induced Ca2+ transient by preincubation with IV.3 Fab. Also, preincubation of PMN with unlabeled mAb 3G8 F(ab')2 did not alter the biotinylated IV.3-Fab + streptavidin-induced rise in [Ca2+] but did block the mAb 3G8 F(ab')2-biotin-induced [Ca2+] transient (Fig. 2C). These results categorically demonstrate that homotypic cross-linking of Fcgamma RIIIb results in a rise in [Ca2+] that is independent of ligand-mediated interactions with the transmembrane Fcgamma RIIa.

Differential Regulation of the Fcgamma RIIa and Fcgamma RIIIb Ca2+ Transients-- Cross-linking of neutrophil Fcgamma R results in tyrosine kinase activity (3, 15, 22). The dependence of the Fcgamma RIIIb-induced Ca2+ transient on protein-tyrosine kinase activity was shown with the tyrosine kinase inhibitor methyl 2,5-dihydroxycinnamate (100 µM), a stable erbstatin analog; the Fcgamma RIIa- and Fcgamma RIIIb-mediated rise in [Ca2+] 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% cells viable). Comparable results were obtained with the tyrosine kinase inhibitors tyrophostin (40 µg/ml, n = 3), lavendustin A (50 µg/ml, n = 2), 2-hydroxy-5-(2,5-dihydroxybenzyl)aminobenzoic acid (1 µg/ml, n = 3), and staurosporine, which inhibits both tyrosine and ser/thr kinases (0.5 µg/ml, n = 3). However, differential sensitivity to the tyrosine kinase inhibitor genistein was observed for the Fcgamma RII- and Fcgamma RIII-mediated [Ca2+] transients. When neutrophils were incubated with 100 µM genistein for 5 min, the rise in [Ca2+] induced by cross-linking Fcgamma RIIa (Fig. 3) and by fMLP (results not shown) was completely abolished. Surprisingly, the Fcgamma RIIIb-induced rise in [Ca2+] was not abrogated by 100 µM genistein (Fig. 3). In four independent paired experiments, the ability of Fcgamma RIIa but not Fcgamma RIIIb to initiate a rise in [Ca2+] was abrogated by 100 µM genistein (Fcgamma RIIa/Fcgamma RIIIb % control, 5 ± 4%/52 ± 6%; n = 4, p < 0.001). The differential sensitivity to genistein was also observed at 50 µM genistein (Fcgamma RIIa/Fcgamma RIIIb % control, 26 ± 3%/63 ± 2%, n = 3, p < 0.001). These concentrations of genistein have been shown to completely inhibit neutrophil Fcgamma RIIa-induced tyrosine phosphorylation and phagocytosis (37, 38) and to block neutrophil degranulation and superoxide production in response to cross-linking of Fcalpha R and L-selectin (35, 36). Little or no sensitivity of the Fcgamma RIIa- or Fcgamma RIIIb-induced Ca2+ transient to the tyrosine kinase inhibitor reduced carboxamidomethylated and maleylated-lysozyme (100 µg/ml, n = 2) or the ser/thr kinase inhibitors calphostin C, H-7, H-8, and H-1004 was observed. These results demonstrate that both Fcgamma RIIa and Fcgamma RIIIb initiate Ca2+ 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.


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  		Fig. 3.   Differential sensitivity of the Fcgamma RIIa- and Fcgamma RIIIb-induced Ca2+ transient to inhibition by the protein-tyrosine kinase inhibitor genistein. Cells were opsonized with the indicated anti-Fcgamma R mAb as described under "Experimental Procedures." After one wash, cells were resuspended in buffer containing Ca2+/Mg2+ and genistein, or methyl 2,5-dihydroxycinnamate was added. After a 5-minute incubation at 37 °C, data acquisition was initiated. F(ab')2 GAM was added as stimulus at 60 s. A representative experiment of three is shown.

It has been shown that neutrophil Fcgamma R-induced superoxide production and phagocytosis are inhibited by occupancy of the adenosine A2 receptor (39-41). Because these responses are dependent on a rise in [Ca2+], we tested the susceptibility of Fcgamma R-specific [Ca2+] transients to the potent adenosine A2 receptor agonist NECA. Neutrophils were treated with varying concentrations of NECA for 5 min at 37 °C before Fcgamma R cross-linking. In the presence of 10-6 M NECA (the same concentration required for inhibition of Fcgamma R phagocytosis (39)), the rise in [Ca2+] induced by cross-linking of Fcgamma RIIa was significantly inhibited (38.0 ± 4.3% of control, n = 4, p < 0.01) (Fig. 4A). In contrast, the Fcgamma RIIIb-mediated rise in [Ca2+] was unaltered by the same concentrations of NECA (98.1 ± 21.5% of control, n = 4, p > 0.05), demonstrating differential regulation of Fcgamma RII and Fcgamma RIII signaling by this adenosine A2 agonist (Fcgamma RIIa versus Fcgamma RIIIb, n = 4, p < 0.01).


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  		Fig. 4.   Differential sensitivity of the Fcgamma RIIa- and Fcgamma RIIIb-induced Ca2+ transient to agents that increase intracellular [cAMP]. A, cells were prepared as described in Fig. 3 except that 10-6 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 Bt2cAMP 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 10-6 M MP and 10-4 M isobutylmethylxanthine was included during the final 5-min preincubation before data acquisition. A representative experiment of six is shown. IBMX, isobutylmethylxanthine.

Engagement of adenosine A2 receptors has been shown to lead to transient increases in intracellular levels of cAMP, increases in intracellular [Ca2+] in mast cells, and to activation of a membrane-associated serine/threonine protein phosphatase (42-44). The ability of NECA to activate PLCgamma and increase [Ca2+] may be the basis for the inhibition of the Fcgamma RIIa-induced Ca2+ transient (cf. Fig. 4A). However, in human neutrophils, the magnitude of the NECA-induced Ca2+ transient is only a fraction of the level of the Fcgamma RIIa-induced transient (40 ± 6 nM rise in Ca2+ after stimulation with 10-6 M NECA, n = 3). Alternatively, it is known that inhibition of Fcgamma R phagocytosis by adenosine A2 receptors is mediated at least in part by increases in intracellular levels of cAMP (40). To determine if the Fcgamma RIIa-induced rise in [Ca2+] is sensitive to increases in cAMP, neutrophils were treated with dibutyryl-cAMP (Bt2cAMP), and a dose-dependent decrease in the Fcgamma RIIa-induced rise in [Ca2+] was observed (at 5 × 10-4 M Bt2cAMP treated versus control, p < 0.02, n = 5) (Fig. 4B). The Fcgamma RIIIb-mediated rise in [Ca2+] was unaffected (5 × 10-4 M Bt2cAMP versus control, p > 0.05, n = 4) (Fig. 4B). Likewise, a prostaglandin E1 analog (MP), which also induces a transient increase in intracellular [cAMP] (45), markedly diminished the Fcgamma RIIa-induced rise in [Ca2+] in the presence of the cAMP phosphodiesterase inhibitor isobutylmethylxanthine (10-4 M) (10-5 M MP + isobutylmethylxanthine versus control, p < 0.001, n = 6), whereas the Fcgamma RIIIb-mediated Ca2+ flux was unaffected (10-5 M MP + isobutylmethylxanthine versus control, p > 0.05, n = 5). (Fig. 4C). Taken together, the marked inhibition of the Fcgamma RIIa-induced Ca2+ transient by NECA, MP, and Bt2cAMP demonstrated that Ca2+ flux induced by Fcgamma RIIa, but not Fcgamma RIIIb, is down-regulated by elevations in intracellular [cAMP].

IP3-dependent and -independent Ca2+ Transients-- Elevations in [cAMP] can induce activation of the cAMP-dependent protein kinase, which in turn can down-modulate PLCgamma 1 activity (46). The sensitivity of the Fcgamma RIIa-induced, but not the Fcgamma RIIIb-induced, rise in [Ca2+] to cAMP suggests that Fcgamma RIIa may engage an IP3-dependent mechanism. Upon maximal homotypic Fcgamma RIIIb cross-linking with saturating levels of mAb 3G8 F(ab')2 and F(ab')2 GAM, there was no detectable increase in IP3 levels, which is in marked distinction to the time-dependent increase in IP3 observed after homotypic cross-linking of Fcgamma RIIa with mAb Fab and F(ab')2 GAM (Table I). As a positive control, the fMLP-induced increase in IP3 levels is shown (Table I).

                              
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Table I
Quantification of PMN IP3 levels after homotypic Fcgamma R cross-linking or fMLP stimulation
Neutrophils were prepared and stimulated with fMLP or the indicated mAb (IV.3 Fab, 0.5 µg/ml; 3G8 F(ab')2, 2 µg/ml; and F(ab')2 GAM, 35 µg/ml) for varying periods of time. Cells were rapidly pelleted, and IP3 levels were determined in cell extracts using a competitive IP3 receptor binding assay as described under "Experimental Procedures." Values represent mean ± S.D. (n = 5).

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(ab')2 using a biotin-avidin coupling technique. The ability of these probes (E-IV.3 Fab and E-3G8 F(ab')2 for Fcgamma RII and Fcgamma RIII, respectively) to cross-link their respective receptors and elicit a rise in [Ca2+] was confirmed by single cell analysis of fura-2-loaded neutrophils (Fig. 5). However, using the mAb-coated E as a stimulus, engagement of Fcgamma RIIa but not Fcgamma RIIIb induced an increase in [IP3] (results not shown).


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  		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.

Biochemical Characterization of the Heterotypic Fcgamma RII+Fcgamma RIII Ca2+ Transient-- Our results with receptor-specific IP3 data do not provide an explanation for the vigorous IP3 response that has been reported during antibody opsonized erythrocyte (EA) phagocytosis (47). EA can engage both Fcgamma RIIa and Fcgamma RIIIb, resulting in heterotypic cross-linking of these receptors. Since heterotypic cross-linking of Fcgamma RIIa and Fcgamma RIIIb results in a synergistic phagocytic response (22) and since neutrophil Fcgamma R phagocytosis is dependent on a receptor-induced rise in [Ca2+], we determined if heterotypic cross-linking of Fcgamma RIIa and Fcgamma RIIIb results in a synergistic [Ca2+] response. Neutrophils opsonized with equivalent densities of either anti-Fcgamma RII mAb IV.3 Fab, anti-Fcgamma RIII mAb 3G8 F(ab')2, or both IV.3 Fab and 3G8 F(ab')2 (verified by flow cytometric analysis) displayed markedly different quantitative [Ca2+] responses with heterotypic cross-linking, showing a synergistic rise in [Ca2+] (Fig. 6).


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  		Fig. 6.   Fcgamma RIIa and Fcgamma RIIIb heterotypic cross-linking induces a synergistic Ca2+ response. Cells were opsonized with the indicated levels of mAb (B) and the GAM-induced Ca2+ response was measured (A). To achieve identical mAb opsonization densities, mAb IV.3 Fab was used at saturation (0.5 µg/ml) for Fcgamma RIIa homotypic cross-linking, mAb 3G8 F(ab')2 was used at a subsaturating dose (0.3 µg/ml) for Fcgamma RIIIb homotypic cross-linking, and the heterotypic cross-linking was induced by using 0.25 µg/ml mAb IV.3 Fab and 0.15 µg/ml mAb 3G8 F(ab')2. A representative experiment of five is shown.

To determine if the heterotypic Fcgamma R cross-linking results in an IP3 response that is distinct from that elicited by homotypic receptor cross-linking, IP3 responses were quantitated after heterotypic cross-linking of Fcgamma RIIa and Fcgamma RIIIb by 3G8 IgG (to avoid pre-opsonization, which can significantly increase base-line IP3 levels (Table I)). In marked contrast to homotypic cross-linking of either receptor, heterotypic Fcgamma R cross-linking (in a Fcgamma RIIa-R131/R131 donor) resulted in a significantly enhanced IP3. In fact, the time-dependent increase in IP3 is very similar to the fMLP response both temporally and in magnitude and more than 2-fold higher than the maximal homotypic Fcgamma RIIa response (Fig. 7A, Table I). In parallel with the synergistic IP3 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/106 cells, in agreement with the range reported in the literature (48), and increased 2-3-fold upon stimulation with heterotypic Fcgamma R cross-linking or fMLP. A comparable increase in diacylglyceride levels was also detected in mAb 3G8 IgG-stimulated cells (21.4 ± 4.3, 58.2 ± 3.9, 73.2 ± 2.1, 41.2 ± 11.4 pmol/106 cells at t = 0, 1, 5, and 10 min, respectively (n = 3)). Homotypic receptor cross-linking did not result in any detectable increase in diglyceride levels. These data show that heterotypic Fcgamma R cross-linking results in a synergistic Ca2+ response that is due at least in part to the synergistic IP3/diglyceride response.


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  		Fig. 7.   Synergistic Fcgamma RIIa-Fcgamma RIIIb-induced production of IP3 and diglyceride. PMN were stimulated with mAb 3G8 IgG (2 µg/ml) or fMLP (10-7 M) for the indicated periods of time, the reactions were terminated at the indicated times by rapid sedimentation, the cells were solubilized, and the [IP3] or [diglyceride] was determined as described under "Experimental Procedures." Values represent the mean ± S.E. (n = 3).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Neutrophil Fcgamma RIIIb elicits a rise in [Ca2+]i that is independent of ligand engagement of Fcgamma RIIa. Like the Fcgamma RIIa-induced change in [Ca2+]i, the Fcgamma RIIIb-induced [Ca2+]i transient is dependent on tyrosine phosphorylation events. However, unlike Fcgamma RIIa, the Fcgamma RIIIb-induced [Ca2+]i transient is resistant to the protein-tyrosine kinase inhibitor genistein, resistant to elevations in [cAMP], and independent of demonstrable changes in [IP3]. These data demonstrate that the biochemical regulation of Fcgamma RIIIb function is distinct from Fcgamma RIIa, and they provide the initial basis for understanding the distinct repertoire of cell programs initiated by Fcgamma RIIIb.

Fcgamma 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 Fcgamma RIIa and SH2 domains in the kinases (3, 12). One characteristic of Syk activation is the tyrosine phosphorylation and activation of PLCgamma , resulting in the breakdown of phosphatidylinositol 4,5-bisphosphate into IP3 and diacylglyceride (49, 50). Indeed, in myelomonocytic cell lines, cross-linking of Fcgamma RIIa is associated with a rapid rise in [IP3] (11, 12). Data suggesting that stimulation of Fcgamma R in human neutrophils does not lead to any change in the intracellular [IP3] (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 IP3 levels, which avoids the biosynthetic labeling of cells with myo-[3H]inositol, a process that is inherently inefficient and difficult to perform in neutrophils due to the necessarily short labeling periods.

Heterotypic neutrophil Fcgamma R cross-linking during Fcgamma R-mediated phagocytosis or immune complex-induced Fcgamma R-stimulation, elicits an IP3 burst (47, 52). Our data indicate that the nature of the Fcgamma R stimulus is critical; there is no detectable increase in IP3 levels after homotypic cross-linking of Fcgamma RIIIb, a small but detectable increase in [IP3] after cross-linking of Fcgamma RIIa, and a substantial generation of IP3 induced by heterotypic cross-linking of neutrophil Fcgamma R with the IP3 response, comparable in magnitude to the fMLP-induced IP3 response. In parallel with the increased [IP3], 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 Fcgamma RIIa and Fcgamma RIIIb in the generation of the early Ca2+ transient (Fig. 6) (53) and perhaps for phagocytosis (22) and the oxidative burst (23). Since cross-linking of Fcgamma RIIIb results in tyrosine phosphorylation of Fcgamma RIIa (22), enhanced tyrosine phosphorylation of Fcgamma RIIa might enhance the ability of this receptor to activate downstream effector molecules such as PLCgamma , leading to the IP3 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 Fcgamma RIIIb co-precipitates in detergent-insoluble domains with hck, which is in contrast to the association of neutrophil Fcgamma RIIa with fgr (15). Differential association and activation of src-kinases by neutrophil Fcgamma RIIa and Fcgamma 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 Fcgamma RIIIb, which plays an important role in anti-neutrophil cytoplasmic antibodies-positive Wegener's granulomatosis (21, 54), might allow targeted down-modulation of neutrophil-mediated injury mechanisms in that disease.

Although our results clearly show that Fcgamma RIIa does induce IP3 production, Fcgamma RIIIb does not elicit an IP3 response after homotypic cross-linking (Table I). This lack of detectable IP3 cannot be due to a lack of sensitivity, since homotypic engagement of Fcgamma RIIIb at receptor saturation consistently results in a Ca2+ transient that is higher in magnitude than the response elicited by homotypic cross-linking of Fcgamma RIIa. Nonetheless, the Fcgamma RIIIb-induced Ca2+ is released from intracellular stores (18). The nature of the intracellular Ca2+-mobilizing signal has yet to be elucidated. Among the IP3-independent mechanisms, cyclic ADP-ribose and sphingosine-1-phosphate are candidates for intracellular Ca2+-mobilizing signals (55-57). Of course, it is also possible that Fcgamma RIIa engages both IP3-dependent and IP3-independent Ca2+-releasing mechanisms. Future studies will be needed to resolve the role of IP3, cyclic ADP-ribose, and sphingosine kinase in neutrophil Fcgamma R-mediated Ca2+ transients.

There are significant implications in the finding that Fcgamma RIIIb does not engage an IP3-mediated signaling pathway. Direct interactions between GPI-anchored proteins and src-family kinases provide one possible mechanism for the transmission of intracellular signal generation. Alternatively, GPI-anchored 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 Fcgamma RIa, Fcgamma RIIIa, Fcalpha RI, and Fcepsilon RI (1, 3). The nature and identity of possible Fcgamma 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 Fcgamma RIIIb, leukocyte function antigen-1 (LFA-1), and the urokinase receptor (58-60). However, the lack of an Fcgamma RIIIb-induced increase in IP3 contrasts to the ability of CD11b/CD18 to induce increases in [IP3] after cross-linking (47). Furthermore, the ability of Fcgamma RIIIb to activate CD11b/CD18 for phagocytosis, a function that CD11b/CD18 cannot do alone in resting neutrophils, indicates that all Fcgamma RIIIb signaling cannot be mediated through CD11b/CD18 (22).

Our results also provide the basis for understanding that the results of Fcgamma R engagement on neutrophils will depend on which receptor type(s) are engaged. An IgG2 ligand will selectively and homotypically engage Fcgamma RIIa of the H131 genotype (61, 62). Anti-neutrophil cytoplasmic antibodies may favor engagement of the more highly expressed Fcgamma RIIIb. In contrast, multivalent immune complexes would favor heterotypic cross-linking of Fcgamma RIIa, Fcgamma 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 Fcgamma R-mediated signal transduction. Ultimately, an understanding of these pathways will enable specific modulation of Fcgamma R-initiated inflammatory processes in autoimmune diseases without complete blockade of all Fcgamma R-mediated functions, which may be essential in normal host defense.

    ACKNOWLEDGEMENTS

We thank Dr. Barbara J. Struthers and G. D. Searle for providing the prostaglandin E1 analog misoprostol and Dr. Bruce Rapuano (The Hospital for Special Surgery, New York, NY) for performing the diglyceride determinations. We are also grateful for the advice and discussions with our colleagues at the Hospital for Special Surgery where this work was performed.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants RO1-AR42476 and RO1-AR33062). The flow cytometry core at the Hospital for Special Surgery was supported by National Institutes of Health Grant P60-AR38320.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Div. of Clinical Immunology and Rheumatology, 1900 University Blvd., University of Alabama, Birmingham, AL 35294. Tel.: 205-934-0894; Fax: 205-934-1564; E-mail: jedberg{at}uab.edu.

§ Present address: Dept. of Immunology, University of Washington, Seattle, WA 98195.

Present address: Div. of Rheumatology, Dept. of Medicine, University of Pennsylvania, Philadelphia, PA 19104.

1 The abbreviations used are: Fcgamma R, receptor for the Fc region of IgG; GPI, glycosylphosphatidylinositol; PMN, polymorphonuclear leukocyte; PLC, phospholipase C; IP3, inositol 1,4,5-trisphosphate; PBS, phosphate-buffered saline; E, erythrocyte; GAM, IgG F(ab')2 fragments of polyclonal goat anti-mouse IgG; MP, misoprostol; fMLP, formylmethionylleucylphenylalanine; NECA, 5'-(n-ethylcarboxamido)adenosine; Bt2cAMP, dibutyryl cyclic AMP.

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Top
Abstract
Introduction
Procedures
Results
Discussion
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P. Y. Coxon, M. J. Rane, D. W. Powell, J. B. Klein, and K. R. McLeish
Differential Mitogen-Activated Protein Kinase Stimulation by Fc{gamma} Receptor IIa and Fc{gamma} Receptor IIIb Determines the Activation Phenotype of Human Neutrophils
J. Immunol., June 15, 2000; 164(12): 6530 - 6537.
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F. Y. S. Chuang, M. Sassaroli, and J. C. Unkeless
Convergence of Fc{gamma} Receptor IIA and Fc{gamma} Receptor IIIB Signaling Pathways in Human Neutrophils
J. Immunol., January 1, 2000; 164(1): 350 - 360.
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J. C. Edberg, A. M. F. Yee, D. S. Rakshit, D. J. Chang, J. A. Gokhale, Z. K. Indik, A. D. Schreiber, and R. P. Kimberly
The Cytoplasmic Domain of Human Fcgamma RIa Alters the Functional Properties of the Fcgamma RI{middle dot}gamma -Chain Receptor Complex
J. Biol. Chem., October 15, 1999; 274(42): 30328 - 30333.
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M. Kocher, J. C. Edberg, H. B. Fleit, and R. P. Kimberly
Antineutrophil Cytoplasmic Antibodies Preferentially Engage Fc{gamma}RIIIb on Human Neutrophils
J. Immunol., December 15, 1998; 161(12): 6909 - 6914.
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