©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Ca Dependence of Human Fc Receptor-initiated Phagocytosis (*)

(Received for publication, June 20, 1995; and in revised form, July 24, 1995)

Jeffrey C. Edberg (1) Ching-Tai Lin (2) Dana Lau (1) Jay C. Unkeless (2) Robert P. Kimberly (1)(§)

From the  (1)Cornell University Medical College, Graduate Program in Immunology, The Hospital for Special Surgery, New York, New York 10021 and the (2)Mount Sinai School of Medicine, Department of Biochemistry, City University of New York, New York, New York 10029

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Differing roles for transients in FcR-mediated phagocytosis have been suggested based on the observations that antibody-opsonized erythrocyte phagocytosis by human neutrophils shows a [Ca] dependence, while that by murine macrophages appears [Ca]-independent. To explore whether this difference might reflect different receptor isoforms or different cell types, we studied the [Ca] dependence of receptor-initiated phagocytosis by human FcRIIa and a panel of FcRIIa cytoplasmic domain mutants expressed in murine P388D1 cells and by human FcR endogenously expressed on human neutrophils and monocytes. Wild-type and point mutants of huFcRIIa stably transfected into murine P388D1 cells have different capacities to initiate a [Ca] transient, which are closely correlated with quantitative phagocytosis (r = 0.94, p < 0.0001). Phagocytosis both by huFcRIIa in P388D1 cells and by huFcRIIa endogenously expressed on neutrophils and blood monocytes shows [Ca] dependence. Phagocytosis of antibody-opsonized erythrocytes by neutrophils demonstrated greater susceptibility to [Ca] quenching compared with FcRIIa-specific internalization with E-IV.3, suggesting that the phagocytosis activating property of FcRIIIb in neutrophils also engages a [Ca]-dependent element. In contrast, phagocytosis by human FcRIa, endogenously expressed on blood monocytes, is [Ca]-independent. Despite the importance of a consensus tyrosine activation motif for both receptors, FcRIa and FcRIIa engage at least some distinct signaling elements to initiate phagocytosis. The recognition that both of the phagocytic receptors on murine macrophages and human FcRIa associate with the FcRI -chain, which contains a tyrosine activation motif distinct from that in the FcRIIa cytoplasmic domain, suggests that [Ca]-independent phagocytosis is a property associated with the utilization of -chains by FcR.


INTRODUCTION

Receptors for the Fc region of IgG (FcR) (^1)provide a link between antibody-antigen complexes and cellular-based effector functions and are critical in the regulation of the inflammatory response(1, 2) . Significant structural diversity between the three gene families encoding FcR is observed(1, 2, 3, 4, 5) . Nonetheless, FcR share certain intracellular signaling pathways. The common themes in FcR signaling pathways involve the activation of protein tyrosine kinases followed by a transient rise in intracellular Ca levels. The [Ca] increase is essential for many cellular functions and is required for the phagocyte FcR-induced oxidative burst(6, 7) .

Many lines of evidence in both human and murine systems indicate that tyrosine phosphorylation events are critical for phagocyte FcR functions, including phagocytosis(8, 9, 10) . In addition, in many systems examined, tyrosine kinase activity is required for the receptor-induced rise in [Ca] (presumably through tyrosine phosphorylation of phospholipase C1 and generation of inositol 1,4,5-trisphosphate). However, the role of [Ca] in Fc receptor-mediated phagocytosis has been controversial. For example, work in murine macrophage cell lines suggests that transients in [Ca] are not essential for phagocytosis of antibody-opsonized erythrocytes (EA) (11, 12, 13) . In contrast, phagocytosis of EA by human neutrophils is significantly impaired by chelation of intracellular calcium and abrogation of [Ca] transients(14, 15) . The ability of [Ca]-depleted neutrophils to mediate phagocytosis initiated by other cell surface receptors suggests that the [Ca]-dependent EA phagocytosis by human neutrophils may reflect a particular property of the Fc receptors on these cells(16) .

Indeed, each of the studies of the [Ca]-dependence of Fc receptor mediated phagocytosis has used EA probes that engage all available Fc receptor types and has not systematically distinguished between different cell types or cells derived from different species. Recent data indicate that important species differences do exist for Fc receptors. For example, human FcRIIA and FcRIIIB, expressed on neutrophils, do not have murine homologues(1, 2, 3) . Human FcRIIA stably transfected into the murine macrophage cell line P388D1 mediates receptor-specific phagocytosis but in a [Ca]-dependent fashion(17) . In contrast, murine FcRII (the IIb isoform) does not have a tyrosine activation motif nor does it trigger a [Ca] transient or protein tyrosine phosphorylation(18) . Murine FcRII is also unable to mediate phagocytosis in macrophages in either a [Ca]-dependent or independent fashion(19) . These observations, coupled with the recent data of Stendahl and co-workers (20) that [Ca]storage organelles accumulate at contact sites during phagocytosis in human neutrophils prompted us to reexamine the question of the [Ca] dependence of Fc receptor-mediated phagocytosis by human Fc receptors, endogenously expressed by human cells and stably transfected into the P388D1 murine macrophage cell line.

Our data indicate that human FcRIIa uses [Ca]-dependent elements to mediate receptor-specific phagocytosis and that huFcRIIa point mutants with a varying ability to initiate a [Ca] transient show a closely corresponding variation in quantitative phagocytosis. Human FcRIIa, endogenously expressed on neutrophils and blood monocytes, also shows partial [Ca] dependence for phagocytosis. In contrast, phagocytosis by human FcRIa, endogenously expressed on blood monocytes, is [Ca]-independent. Despite the importance of tyrosine phosphorylation for phagocytosis and the use of a consensus tyrosine activation motif by both receptors(1, 2, 3, 4, 5, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) , they engage at least some distinct signaling elements to initiate phagocytosis. The recognition that the human FcRIa associates with the FcRI -chain, which contains the tyrosine activation motif (21, 22) as do both of the phagocytic receptors on murine macrophages(23) , suggests that [Ca]-independent phagocytosis is a property associated with FcR utilizing -chains. Human FcRIIa, while engaging many elements in common with human FcRIa such as p72 and phospholipase C1(25, 26, 27, 28, 29, 30) , must also engage [Ca]-dependent elements.


MATERIALS AND METHODS

Reagents

NHS-LC-biotin, sulfo-NHS-biotin, and streptavidin were obtained from Pierce. BAPTA-AM and Indo-1/AM were from Molecular Probes (Eugene, OR). A 10 mM stock of BAPTA-AM in dimethyl sulfoxide was prepared and stored at -20 °C. Genistein was from Life Technologies, Inc. and was stored as a 20 mg/ml stock in dimethyl sulfoxide at -20 °C. Anti-FcR mAbs 32.2, 22 (FcRI, CD64) and IV.3 (FcRII, CD32) Fab or F(ab`)(2) fragments were obtained from Medarex (Annandale, NJ); IgM anti-H-2D^d was obtained from Pharmingen (San Diego, CA). Fab and F(ab`)(2) fragments were free of intact IgG as detected by silver stain analysis after SDS-polyacrylamide gel electrophoresis and size exclusion high performance liquid chromatography. Unconjugated F(ab`)(2) goat anti-mouse IgG (GAM) for mAb cross-linking and phycoerythrin- and fluorescein isothiocyanate-conjugated GAM for immunofluorescence flow cytometry studies were obtained from Jackson Immunoresearch (West Grove, PA) or Boehringer Mannheim. Fetal calf serum and RPMI 1640 were obtained from Life Technologies, Inc. All other reagents were from Sigma.

Cells and Cell Lines

Mutant FcRIIA cDNAs were made by oligonucleotide primer-directed site-specific mutagenesis of a human FcRIIA cDNA generously provided by J. Kochan (Hoffman-La Roche, Nutley, NJ)(31) . Mutants, confirmed by sequencing (Sequenase 2.0, U. S. Biochemical Corp.), were subcloned into pcEXV-3 (32) and transfected by CaPO(4) precipitation in the presence of 25-100 µM chloroquine into the murine macrophage cell line, P388D1. Stable transfectants were screened and selected by flow cytometry, and assessment of receptor expression was demonstrated between 1.1 and 2.5 times 10^6 receptors/cell.

Fresh anti-coagulated human peripheral blood was separated by centrifugation through a discontinuous two-step Ficoll-Hypaque gradient (33) . Mixed mononuclear cells were isolated from the upper interface and washed with Hanks' balanced salt solution. Neutrophils were isolated from the lower interface and washed with Hanks' balanced salt solution. Contaminating erythrocytes in the neutrophil harvest were lysed with hypotonic saline (0.2% NaCl) for 20 s followed by 1.6% NaCl and a final wash with Hanks' balanced salt solution. After final washes, cells were resuspended to 5 times 10^6 cells/ml in PBS prior to immunofluorescent staining or in RPMI containing 10% fetal calf serum prior to phagocytosis.

Treatment of cells with BAPTA (to quench intracellular Ca levels) or genistein (to block protein tyrosine kinase activity) was performed as described previously(10, 17, 34) . Briefly, cells were incubated with BAPTA-AM (1-100 µM) in buffer without free Ca for 30 min at room temperature (neutrophils and monocytes) or 37 °C (P388D1 transfectants) followed by one wash. Buffer containing Ca was then added and handled as described below. For genistein treatment, cells were preincubated with 100 µg/ml genistein for 30 min, and then the genistein was maintained at the same concentration through the phagocytic assay. Controls included loading cells with the BAPTA-AM and genistein solvent (dimethyl sulfoxide) at appropriate concentrations for the same period of time. As an alternative to BAPTA-AM treatment for quenching [Ca](i), cells were allowed to drain their intracellular Ca stores as described by Rosales et al.(35) . Levels of intracellular Ca were directly determined in all cases as described below.

Measurements of [Ca](i)

Intracellular [Ca](i) was determined in Indo-1/AM-loaded cells using an SLM 8000 fluorimeter and the simultaneous 405/490 nm fluorescence emission ratio as described previously(17, 34) . Briefly, suspensions of cells at 10^7/ml in Ca- and Mg-free phosphate-buffered saline, pH 7.4, were incubated with 5 µM Indo-1/AM at 37 °C for 15 min and washed in PBS. Cells preparations to be opsonized with receptor-specific mAb Fab were resuspended in Ca-and Mg-free PBS at 10^7 cells/ml, incubated with saturating concentrations of mAb IV.3 Fab (0.5 µg/ml) or mAb 22 F(ab`)(2) (2 µg/ml) at 37 °C for 5 min, and washed in PBS. All cell preparations were resuspended in 1.1 mM Ca, 1.6 mM Mg PBS at 37 °C for 5 min and then immediately transferred to a continuously stirred cell cuvette maintained at 37 °C in the SLM 8000. With excitation at 355 nm, the simultaneous fluorescence emission at 405 and 490 nm was measured, integrated, and recorded each second. After establishing a base line for 60 s, goat anti-mouse F(ab`)(2) was added (35 µg/ml final concentration), and data acquisition was continued for an additional 3.5 min. Each sample was individually calibrated by lysing cells in 1% Triton X-100 to determine the maximal emission ratio and by adding EDTA (20 mM final concentration) to determine the minimal ratio. The Indo-1 fluorescence emission ratio was converted to [Ca](i) by the method of Grynkiewicz et al.(36) .

Measurement of Phagocytosis

Biotinylated bovine erythrocytes (E(B)) and biotinylated anti-FcR were prepared as described previously(37) . Briefly, erythrocytes (at 1 times 10^9 cells/ml in 0.1 M carbonate buffer (pH 8.6)) were incubated with 250 µg/ml of sulfo-NHS-biotin for 20 min at 4 °C with mixing. E(B) (1 times 10^9 E/ml) were coated with an equal volume of streptavidin (250 µg/ml) for 30 min at 4 °C with mixing. The streptavidin-coated E(B) (E) were then washed and resuspended to 1 times 10^9 erythrocytes/ml for immediate use. mAb were biotinylated with NHS-LC-biotin in 0.1 M carbonate buffer (pH 8.6). Typically, 50 µg/ml NHS-LC-biotin was used to biotinylate mAb (1 mg/ml) for 60 min at room temperature with occasional mixing. The free biotin was removed by extensive dialysis against PBS (pH 7.4). Small volume dialysis (ranging from 50-100 µl for the mAb) was performed in a dialysis chamber (Pierce).

mAb-conjugated erythrocytes were prepared by incubating E with dilutions of biotinylated anti-FcR mAb(37) . mAb-coated E were resuspended in RPMI 1640-fetal calf serum, an aliquot was removed for analysis by indirect immunofluorescence, and the remaining cells were used immediately for the phagocytosis or attachment assays. EA were prepared by incubating bovine erythrocytes with a 1:4 dilution of the maximal subagglutinating titer of rabbit anti-bovine erythrocyte IgG as described previously(38) .

For quantitation of mAb-coated E or EA phagocytosis by fresh human cells, erythrocytes were mixed with 100 µl of fresh neutrophils (5 times 10^6 cells/ml) or fresh mononuclear cells (5 times 10^6 monocytes/ml, determined by myeloperoxidase staining) at a ratio of 25:1 (mAb-coated E) or 50:1 (EA) (37) . The cell mixture was pelleted for 5 min at room temperature at 44 times g and then incubated at 37 °C for 20 min (neutrophils) or 1 h (mononuclear cells). After the nonphagocytosed erythrocytes were lysed with hypotonic saline (0.2% NaCl for 20 s followed by the addition of an equal volume of 1.6% NaCl), phagocytosis was quantitated by light microscopy. The data are expressed as phagocytic index (PI, the number of ingested particles/100 neutrophils or monocytes).

Phagocytosis by transfected P388D1 cells was determined in an adherent assay system. P388D1 cells (5 times 10^5 cells/ml) were allowed to adhere to round glass coverslips in culture dishes overnight at 37 °C. Coverslips were then transferred to clean culture dishes and EA- or mAb-coated E were added (50 µl at 5 times 10^7 erythrocytes/ml) were added and incubated for 1 h at 37 °C. Noninternalized erythrocytes were lysed by brief immersion of the coverslip in dH(2)O followed by immersion in buffer. Phagocytosis was quantitated by light microscopy and expressed as phagocytic index as described above.

Heat-treated and serum-treated zymosan were prepared as described previously(38) . Briefly, heat-treated zymosan were prepared by boiling 10 mg of zymosan for 10 min. Serum-treated zymosan were prepared by incubating 2 mg of zymosan with 2 ml of normal human serum for 30 min at 37 °C. Following washing, both heat-treated zymosan and serum-treated zymosan were resuspended to 2.5 times 108/ml. For phagocytosis, heat-treated zymosan or serum-treated zymosan were mixed with neutrophils (5:1, zymosan/neutrophil ratio), pelleted, and incubated for 20 min at 37 °C. Phagocytosis was assessed by light microscopy.

Determination of Fc Receptor Alleles

Determination of FcRIIIb alleles, NA1 and NA2, was performed by quantitative flow cytometry with mAbs CLB-FcR-gran 1, CLB-gran 11, and GRM1(33, 38) . The assignment of NA type was confirmed by leukoagglutination as described previously (38) and by immunoprecipitation of selected donors(33) . Phenotyping of donors for the LR-HR alleles of FcRIIa was performed by quantitative flow cytometry using mAbs 41.H16 and IV.3 as described previously(39, 40) .

Data Analysis

Phagocytosis data are displayed as the mean ± S.D. Ca data are representative experiments. Differences in phagocytosis between phagocytic probes were compared with a Student's t test and differences between probes over a range of BAPTA concentrations (see Fig. 4B) was determined using two-way analysis of variance.


Figure 4: A, receptor-specific fluxes in [Ca] were elicited in Indo-1-loaded neutrophils by cross-linking FcRIIa with mAb IV.3 Fab and GAM F(ab`)(2). Pretreatment of cells with increasing concentrations of BAPTA-AM led to a progressive decline in the [Ca] response. Each sample was calibrated as described by Grynkiewicz et al.(35) . A representative experiment is shown (n = 3). B, in parallel experiments, neutrophils were assayed for phagocytosis of E-IV.3 by FcRII (bullet), of rabbit antibody opsonized erythrocytes (EA), which engage both FcRIIa and FcRIIIb (circle), and of heat-treated and serum-treated zymosan (up triangle, box, respectively). Maximal inhibition of both E-IV.3 and EA phagocytosis was reached by 50 µM BAPTA, while phagocytosis of the zymosan particles was unaffected by the same dose of BAPTA. The untreated control PI for EA of 33 was not different from the untreated control PI for E-IV.3 of 29 (p = not significant). The means and standard deviations are shown, and each point represents 5-15 independent determinations.




RESULTS

Several recent observations including information on species differences in Fc receptor isoforms and function (1, 2, 17, 19) have prompted a reconsideration of the studies supporting [Ca](i)-dependent and [Ca](i)-independent Fc receptor-mediated phagocytosis. For example, studies by Stendahl et al.(20) have shown that there is an accumulation of [Ca](i) storage organelles during phagocytosis in human neutrophils. These observations suggest the possibility that the Fc receptors expressed in human neutrophils are functionally distinct from murine Fc receptors in engaging [Ca](i)-dependent elements for phagocytosis. Indeed, initial studies of human FcRIIA truncation mutants stably transfected into P388D1 cells have shown that all truncations unable to initiate a [Ca](i) transient are unable to mediate receptor-specific phagocytosis(17) . The evidence for an essential role for [Ca](i) in FcRIIa phagocytosis is strengthened by the ability of BAPTA, a chelator of [Ca](i), to block phagocytosis by FcRIIa wild-type receptor in P388D1 cells(17) . Accordingly, we have examined these relationships in a series of FcRIIa transfectants with point mutations in the region of the cytoplasmic domain containing the YXXL tyrosine activation motif. Mutations in this region (Fig. 1) can lead to altered binding and activation of p72, which in turn phosphorylates phospholipase C-1 leading to the generation of inositol 1,4,5-trisphosphate and [Ca](i) transients(41) . Studies of transfected FcR mutants indicate that receptor-mediated phagocytosis is also altered (42, 43


Figure 1: Comparison of the tyrosine activation consensus motif (46) with that for human FcRIIA and human -chain of FcRI shows that FcRIIA has a 12-residue, rather than a 7-residue, sequence between the two YXXL motifs (A). Point mutations were designed to alter both the YXXL motifs and adjacent residues (B).



Fifteen mutants were constructed (Fig. 1), and stable transfectants expressing between 1.1 and 2.6 times 10^6 receptors/cell were selected. The [Ca] transient observed after cross-linking each mutant receptor was measured and ranged from no response for several mutants of tyrosine residues within the tyrosine activation motif to a flux of approximately 500 nM (Fig. 2). The measured [Ca](i) transients were abrogated by pretreatment of cells with BAPTA, were unaffected by 10 mM EGTA extracellularly, and therefore were due to mobilization of [Ca](i) from intracellular stores. Among the 15 cell lines expressing different mutant FcRIIa, there was no significant relationship between quantitative receptor expression measured by flow cytometry and peak [Ca](i) flux (p > 0.10; not significant).


Figure 2: A , receptor-specific fluxes in [Ca] were initiated in Indo-1-loaded transfected P388D1 cells by cross-linking human FcRIIa with mAb IV.3 Fab and GAM F(ab`)(2) as described previously in selected mutant FcRIIa expressing cells. Each sample was calibrated as described by Grynkiewicz et al.(36) . B, receptor-specific phagocytosis in selected mutated FcRIIa transfected P388D1 cells was determined using mAb IV.3 Fab coated erythrocytes as described previously(37) . The mean and the standard deviation are shown (n = 6 for each mutant).



The same mutants were probed for FcRIIa-specific phagocytosis using the receptor-specific mAb IV.3 Fab conjugated to erythrocytes via a streptavidin bridge (E-IV.3). The density of mAb IV.3 conjugation to erythrocytes was monitored by flow cytometry. Nonbiotinylated erythrocytes and biotinylated but unconjugated erythrocytes neither bound nor were internalized by transfected or nontransfected P388D1 cells. Furthermore, erythrocytes coupled to an IgM anti-H2-D^d via the streptavidin bridge bound to P388D1 as expected but were not internalized (mean attachment index = 133; phagocytic index = 0; n = 3). Quantitative phagocytosis of E-IV.3 ranged from no internalization for the same tyrosine mutants that failed to elicit a [Ca](i) transient to a maximum phagocytic index of 126.5 ± 16 E-IV.3 ingested per 100 cells (Fig. 2B). Among the 15 different mutants, there was no significant relationship between quantitative receptor expression and E-IV.3 ingestion (p > 0.10; not significant). As with wild-type FcRIIa(17) , the protein tyrosine kinase inhibitor genistein inhibited by >95% phagocytosis of E-IV.3 by the phagocytic mutant forms of FcRIIa and of EA by the native murine FcR in parental P388D1.

There was, however, a striking relationship between peak [Ca](i) and FcRIIa-specific phagocytosis (Fig. 3) with a correlation coefficient of 0.94 (p < 0.0001). Importantly, even the most phagocytic of the mutant receptors was sensitive to chelation of [Ca](i) by 50 µM BAPTA (Fig. 3, inset).


Figure 3: Peak [Ca](i) flux, as demonstrated in Fig. 2, was strongly correlated with quantitative, receptor-specific phagocytosis measured with E-IV.3 (correlation coefficient = 0.94; p < 0.0001). Of note, the EE239-240QQ mutant, which is located in a region outside of the YXXL dyad and has a high [Ca] flux occurs within the 7 residues defined as essential for Delta[Ca] by Kolanus et al.(43) . Inset, pretreatment of phagocytic cells with 50 µM BAPTA-AM-abrogated phagocytosis. A representative experiment is shown (n = 3 for each determination).



The strong correlation between [Ca](i) and phagocytosis for human FcRIIa even in the environment of a murine macrophage cell line and the dependence of FcRIIa-mediated phagocytosis on [Ca](i) suggested that this property might reflect the characteristics of human FcRIIa per se. Since previous studies in human and murine cells had not probed Fc receptor function in a receptor-specific fashion(11, 12, 13, 14, 15) , we sought to explore the properties of FcRIIa expressed endogenously on neutrophils. Our previous studies with neutrophils indicated that FcRIIa alone can mediate phagocytosis(10) . Therefore, using receptor-specific engagement of FcRIIa with mAb IV.3 Fab and cross-linking with GAM F(ab`)(2), we defined the ability of BAPTA pretreatment of neutrophils to blunt the [Ca](i) response (Fig. 4A). Correspondingly, we assessed quantitative receptor-specific phagocytosis (Fig. 4B). A BAPTA dose-dependent inhibition of the FcRIIa-mediated rise in [Ca](i) was observed with complete inhibition achieved by 50 µM BAPTA. BAPTA also reduced phagocytosis in a dose-dependent fashion with 50% reduction at a loading concentration of 50 µM (p < 0.002). Neither phagocytosis of heat-treated zymosan nor of serum-treated zymosan was altered by 50 µM BAPTA pretreatment (both 99-100% of untreated control cells; p > 0.5, not significant) (Fig. 4B). Higher loading concentrations of BAPTA did not lead to further decrement of E-IV.3 internalization (PI = 50.6% of control at 100 µM). 50 µM BAPTA was also sufficient to abrogate detectable [Ca](i) transients initiated by 10 FMLP (Delta[Ca](i) indistinguishable from base line, n = 7).

As an alternative technique to deplete intracellular free Ca levels, we allowed neutrophils to incubate in Ca/Mg-free media to exhaust intracellular Ca stores (Fig. 5) as described by Rosales and Brown(35) . FcRIIa-specific phagocytosis by neutrophils treated in this manner was markedly blunted relative to control cells in the presence of physiologic levels of Ca/Mg (PI = 48.0 ± 13.1% of control, n = 3). The extent of inhibition was comparable with that achieved by BAPTA pretreatment.


Figure 5: Incubation of neutrophils in Ca/Mg-free buffer empties intracellular Ca stores. Addition of FMLP (10M) results in a rapid rise in [Ca] in control neutrophils (neutrophils that were Ca depleted and then repleted), and this response is completely blunted in Ca-depleted cells. A representative experiment is shown (n = 4).



To compare our results in neutrophils with those of Lew and co-workers (14) , we also probed neutrophils with EA for the effects of [Ca](i) chelation on phagocytosis engaging both FcRIIa and FcRIIIb. Although the GPI-anchored FcRIIIb does not mediate phagocytosis itself, it does elicit a [Ca](i) transient and functions synergistically with FcRIIa for an enhanced phagocytic response(10) . As reported by Lew(14) , EA phagocytosis was profoundly reduced by [Ca](i) chelation (Fig. 4B) and to a significantly greater extent than E-IV.3 (EA = 23 ± 15% (n = 10) of control compared 47 ± 20% (n = 12) of control for E-IV.3 at 50 µM BAPTA, p < 0.005; over all doses of BAPTA, EA versus E-IV3, two-way analysis of variance: F = 13.2, p < 0.0002). These observations suggested that the FcRIIIb-initiated [Ca](i) transients might play an important quantitative role in FcRIIa internalization.

To test this hypothesis as the basis for the known difference in EA phagocytosis by individuals homozygous for the two different alleles of FcRIIIb(38, 40) , we examined the relative ability of FcRIIIb in NA1 and NA2 homozygotes to elicit [Ca](i) fluxes in neutrophils. Engagement of FcRIIIb by anti-receptor mAb 3G8 and cross-linking with either GAM or with streptavidin leads to a [Ca](i) flux derived from intracellular stores (34) . When the anti-FcRIII mAb 3G8 IgG, a murine IgG1, was used to initiate the [Ca](i) transient, consistent differences between donors were noted that were attributable to the His-131/Arg-131 polymorphism of FcRIIa and presumably the ability of FcRIIa to engage the Fc region of 3G8 IgG and form heterotypic FcRIIa-FcRIIIb receptor clusters (Fig. 6A). However, no consistent difference in the magnitude of the [Ca](i) flux could be attributed to the phenotype of FcRIIIb engaged by 3G8 F(ab`)(2) with subsequent cross-linking in five matched pairs of NA homozygous donors (Fig. 6B). Taken together, these observations suggest that a [Ca](i)-sensitive signaling element is engaged by FcRIIa during EA phagocytosis but that the magnitude of the FcRIIIb-induced [Ca](i) flux per se does not explain the difference in quantitative phagocytosis between NA1 and NA2 homozygous donors.


Figure 6: A, the ability of intact mAb 3G8 IgG to elicit a [Ca]flux in Indo-1-loaded neutrophils was assessed in a series of donor pairs. A consistent difference in quantitative Delta[Ca] was noted between donors homozygous for the FcRIIa His-131 (LR) and Arg-131 (HR) alleles. Homozygous Arg-131 (HR) donors are able to bind murine IgG1 more effectively to form a receptor cluster of FcRIIIb via Fab binding and FcRIIa via Fc binding thereby triggering a more vigorous [Ca] response (n = 6 pairs of donors). B, 3G8 F(ab`)(2), which engages only FcRIIIb, elicits a [Ca] when cross-linked with goat anti-mouse F(ab`)(2), but no difference between NA1 and NA2 homozygotes could be defined (n = 5 pairs of donors examined in at least two independent experiments).



Demonstration of the [Ca](i) dependence of E-IV.3 phagocytosis in neutrophils and in murine P388D1 cells resolves the apparent controversy between DiVirgilio and others about the role of [Ca](i) in phagocytosis(11, 12, 13, 14, 15) , but it does not address the issue of whether a human homologue of a ``[Ca](i)-independent'' murine receptor would also be [Ca](i) independent for phagocytosis. Accordingly, we examined the effects of BAPTA on FcRIIa-specific (E-IV.3) and FcRIa-specific (E-22) phagocytosis by human peripheral blood monocytes. Both receptors mediate [Ca](i) transients after receptor cross-linking that are blocked by 50 µM BAPTA (Fig. 7A). As anticipated, E-IV.3 showed more than a 50% decrement in phagocytosis after pretreatment with 50 µM BAPTA (Fig. 7B; PI = 40.5 ± 13.5% of control, p < 0.002). In contrast E-22, the specific probe for FcRIa, showed only a minimal change that was significantly less than E-IV.3 (p < 0.002) and not significantly different from control (Fig. 7B, p > 0.5). These data for human FcRIa are similar to those for phagocytic murine Fc receptors on elicited peritoneal macrophages(12, 44) .


Figure 7: A, both FcRIIa and FcRIa can initiate a [Ca] flux in human blood monocytes with cross-linking of receptors, which is completely blocked with 50 µM BAPTA (n = 3). B, but only FcRIIa-specific phagocytosis is significantly affected by pretreatment of cells with 50 µM BAPTA-AM (mean and standard deviation is shown, n = 4). *, p < 0.002, 50 µM BAPTA treated versus control.




DISCUSSION

Recent observations have prompted a re-evaluation of the role of [Ca](i) in Fc receptor-mediated phagocytosis. In previous work, both murine macrophage cell lines and human neutrophils were used, which may have confused the issues since the cells express structurally distinct Fc receptors and may have intrinsically different signaling capacities and cell programs. Indeed, the two Fc receptors constitutively expressed on human neutrophils do not have corresponding murine homologues. While human FcRIIa is phagocytic, murine FcRII (the FcRIIb isoform) on macrophages is unable to mediate [Ca] transients, induction of tyrosine phosphorylation, or phagocytosis(18, 19) . These observations, coupled with the [Ca](i) dependence of huFcRIIa-mediated phagocytosis in stably transfected cells, suggest that the apparent controversy might simply reflect structurally different receptors each engaging at least some distinct signaling elements.

Our data indicate that human FcRIIa, both in a murine macrophage environment and endogenously expressed in human neutrophils, shows significant [Ca](i) dependence for phagocytosis. This is in contrast to human FcRIa, which like its murine homologue, shows minimal or no [Ca] dependence. These observations emphasize that FcRIIa, which has a cytoplasmic tyrosine activation motif distinct from that in the -chain associated with FcRIa (due to 12 versus 7 amino acids separating the YXXL sequences, respectively), engages some signaling elements distinct from FcRIa. The fact that FcRIIa phagocytosis in native cells was not completely [Ca](i)-dependent suggests, however, that this receptor may engage several signaling pathways, a property now recognized for other Fc receptors(45) .

The mechanism underlying the variation in [Ca](i) transients and phagocytosis for different FcRIIa mutants may relate to variable efficiency in engaging the SH2 domain of the protein tyrosine kinase p72. FcRIIa does bind p72(25, 26, 28, 29) , and the binding of the ZAP70 homologue to a similar YXXL tyrosine activation motif shows a high degree of sensitivity to mutations in the flanking sequences (41, 46, 47, 48) . In the FcRI model system, which incorporates the YXXL tyrosine activation motif in -chain, p72 binding and phosphorylation leads to tyrosine phosphorylation of phospholipase C1 (either directly by p72 or through an intermediary kinase(s)), inositol lipid breakdown with generation of inositol 1,4,5-trisphosphate, and a [Ca](i) flux(49, 50, 51, 52, 53) . Disruption of this sequence by inhibition of p72 with piceatannol or specific inhibitory peptide abrogates the [Ca](i) flux(53, 54) . The essential role for [Ca](i) in FcRIIa-mediated phagocytosis is strongly supported by the correlation of [Ca](i) with phagocytosis between the 15 different FcRIIa mutants, by the ability of BAPTA to abrogate phagocytosis by both wild-type and mutant receptors (Fig. 3) and by the ability of BAPTA to abrogate phagocytosis by FcRIIa in neutrophils without affecting ingestion of zymosan and in monocytes without affecting internalization by FcRIa. However, the critical [Ca](i)-dependent element(s) for FcRIIa signaling remain unidentified at present. Preliminary experiments with cyclosporin A, an inhibitor of calcineurin that is a [Ca](i)-dependent phosphatase, show little or no effect on human FcRIIa phagocytosis in transfected P388D1 cells, although calcineurin activity is essential for neutrophil motility in some circumstances(55) . Recent data suggest that L-plastin, a [Ca](i)-regulated actin-bundling protein, may be a candidate for a [Ca](i)-dependent element essential for Fc receptor-mediated phagocytosis in neutrophils (56) .

The greater susceptibility of EA phagocytosis to Ca buffering compared with FcRIIa-specific phagocytosis with E-IV.3 suggests that the activation of phagocytosis by FcRIIIb in human neutrophils has Ca-dependent elements. Since FcRIIIb elicits a [Ca](i) flux and functions synergistically with FcRIIa for phagocytosis, an allele-specific variation in FcRIIIb-initiated [Ca](i) would provide a straightforward mechanism for the quantitative difference in phagocytosis shown by donors homozygous for different FcRIIIb alleles. Although we could define consistent differences in [Ca](i) transients between individuals, with those elicited by intact anti-receptor IgG relating to the His-131/Arg-131 polymorphism of FcRIIa(57, 58, 59) , donors homozygous for the NA1/NA2 FcRIIIb alleles were not different in their ability to generate FcRIIIb-specific fluxes in [Ca](i). Thus, some other mechanism, perhaps relating to the differences in glycosylation of the NA1 and NA2 alleles and potential carbohydrate-mediated interactions with other cell surface molecules such as CD11b/CD18 (60, 61) must be involved. While we cannot explain the NA1/NA2 difference in phagocytosis on the basis of quantitative differences in [Ca](i) interacting with the partially [Ca](i)-dependent phagocytosis of FcRIIa, the difference in [Ca](i) dependence between FcRIIa and FcRIa underscores the fact that different Fc receptors can engage distinct signaling elements. Whether these distinct elements reflect primary sequence differences in the tyrosine activation motifs used by each of the receptors or some modifying contribution of the cytoplasmic domain of the ligand binding FcRIa alpha chain (compare (52) ) remains to be determined. These distinct elements may converge on some cell programs, but they also provide the foundation for differences in elicited cell programs and for selective therapeutic intervention.


FOOTNOTES

*
The work was supported by United States Public Health Service Grants AR-33062 (to R. P. K.), AI-24322, and AI-24671 (to J. C. U.) and a Young Scholar Award from the New York Chapter of the Arthritis Foundation (to J. C. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: The Hospital for Special Surgery, Cornell University Medical Center, 535 E. 70th St., New York, NY 10021. Tel.: 212-606-1214; Fax: 212-717-1192.

(^1)
The abbreviations used are: FcR, receptors for the Fc region of IgG; EA, bovine erythrocytes opsonized with the IgG fraction of rabbit antibovine erythrocyte polyclonal antisera; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid; mAb, monoclonal antibody; GAM, polyclonal F(ab`)(2) goat anti-mouse IgG; PBS, phosphate-buffered saline; PI, phagocytic index (number of erythrocytes phagocytosed per 100 phagocytes); E(B), biotinylated bovine erythrocytes; E, avidin coated E(B); FMLP, formylmethionylleucylphenylalanine; E-IV.3 and E-22, erythrocytes coated with the anti-FcRII mAb IV.3 Fab fragments or the FcRI mAb 22 F(ab`)(2) fragments through a biotin-avidin bridge.


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