J Biol Chem, Vol. 274, Issue 42, 30328-30333, October 15, 1999

The Cytoplasmic Domain of Human FcRIa Alters the Functional Properties of the FcRI·-Chain Receptor Complex^*

Jeffrey C. Edberg^§, Arthur M. F. Yee^¶, Diptendu S. Rakshit^¶, David J. Chang, Jayashree A. Gokhale^¶, Zena K. Indik, Alan D. Schreiber, and Robert P. Kimberly

From the  Departments of Medicine and Microbiology, The University of Alabama at Birmingham, Birmingham, Alabama 35209, the ^¶ Department of Medicine, Hospital for Special Surgery and Weill Medical College of Cornell University, New York, New York 10021, and the  Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The /-chain family of proteins mediate cell activation for multiple immunoglobulin receptors. However, the recognition that these receptors may have distinct biologic functions suggests that additional signaling elements may contribute to functional diversity. We hypothesized that the cytoplasmic domain (CY) of the ligand binding -chain alters the biological properties of the receptor complex. Using macrophage FcRIa as a model system, we created stable transfectants expressing a full-length or a CY deletion mutant of human FcRIa. Both receptors functionally associate with the endogenous murine -chain. However, we have established that the CY of FcRIa directly contributes to the functional properties of the receptor complex. Deletion of the FcRIa CY leads to slower kinetics of receptor-specific phagocytosis and endocytosis as well as lower total phagocytosis despite identical levels of receptor expression. Deletion of the CY also converts the phenotype of calcium independent FcRIa-specific phagocytosis to a calcium-dependent phenotype. Finally, deletion of the CY abrogates FcRIa-specific secretion of interleukin-6 but does not affect production of interleukin-1. These results demonstrate a functional role for the CY of FcRIa and provide a general model for understanding how multiple receptors that utilize the -chain can generate diversity in function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Fc receptors play a central role in the handling of immune complexes, regulation of inflammatory responses, antibody secretion, and T cell activity (1-4). Common to each of these functions is the initiation of tyrosine phosphorylation following receptor cross-linking (5) and the involvement of the / subunits leading to the view that Fc receptors subserve redundant signaling functions. However, recent evidence suggests that these receptors are not redundant. For example, FcRIIIa appears necessary for initiating the Arthus inflammatory reaction (6, 7), while FcRIa and FcRI can down-regulate inflammatory responses by initiating the secretion of IL¹-10 and IL-1ra, respectively (4, 8). The basis for these differences are unknown.

FcRI is expressed on the cell surface in association with the -chain (9, 10). This association is not a prerequisite for transient receptor expression but is necessary for stable expression (11, 12). The -chain cytoplasmic domain contains an immunoreceptor tyrosine activation motif (ITAM) and current data suggest that the -chain cytoplasmic domain is both necessary and sufficient for FcRIa induced functions (13-15). Biochemical studies have shown that cross-linking of the FcRIa·-chain complex results in activation of a Src family kinase(s) and the tyrosine kinase p72^Syk (2, 5). Activation of these kinases results in tyrosine phosphorylation of the -chain and the initiation of a signaling cascade that can culminate in the induction of degranulation, phagocytosis, an oxidative burst, ADCC activity and the induction of gene transcription. The association between FcRIa and -chain may also be important in the formation of a higher affinity receptor complex through the recruitment of two ligand binding chains to the  homodimer (16).

Unlike the -chain, the cytoplasmic domain of FcRI does not contain an ITAM or other tyrosine containing signaling motifs. Nonetheless, murine FcRI on J774 cells is constitutively phosphorylated on serine and after phorbol 12-myristate 13-acetate stimulation the level of phosphorylation increases (17). The cytoplasmic domain of FcRI may also associate with actin-binding protein-280 (ABP-280, also known as non-muscle filamin) in the absence of ligand (18). Receptor engagement by ligand apparently abrogates this association, although its functional significance is not clear. Both of these observations suggest that the cytoplasmic domain of FcRIa may be actively involved in the biologic phenotype of FcRI. Furthermore, FcRIa in the absence of the -chain can signal for calcium in COS-1 cells and the transmission of this calcium signal requires the FcRIa cytoplasmic domain (19). Based on these observations, and the observations that several -chain associated Fc receptors initiate functionally distinct cell programs, we hypothesized that the FcRIa cytoplasmic domain may serve to modify the signaling of the FcRI·-chain receptor complex. By directly comparing wild type human FcRIa with a cytoplasmic domain deletion mutant of FcRIa expressed at comparable levels in stable transfectants of the murine macrophage cell line P388D1, we have established that the cytoplasmic domain of FcRIa alters the functional properties of the receptor complex. These observations provide a general framework for understanding the unique properties of the family of Fc receptors which associate with the -chain.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture and Reagents-- The murine macrophage cell line P388D1 stably transfected with a cDNA encoding human FcRIa or a mutant form of FcRIa containing a stop codon after the first amino acid of the cytoplasmic domain (Lys³¹⁵  Stop 315) were prepared as described previously (13). P388D1 cells transfected with human FcRIIa were previously described (20). All cell lines were maintained as adherent cultures (Corning Tissue Culture Dishes) in RPMI 1640 as described previously (20). All tissue culture reagents were from Life Technologies, Inc. (Grand Island, NY).

Human and mouse IgG were obtained from Sigma. Mouse F(ab')₂ fragments and F(ab')₂ goat anti-mouse IgG (GM) were obtained from Jackson ImmunoResearch (West Grove, PA). F(ab')₂ fragments of the anti-FcRIa mAbs 22.2 and 32.2 were obtained from Medarex (Annandale, NJ). IgM anti-H-2D^d (clone 3-25.4) was obtained from Pharmingen (San Diego, CA). The hybridoma line expressing the rat anti-murine FcRII/FcRIII mAb 2.4G2 was obtained from ATCC (Manassas, VA). All other reagents were from Sigma. Quantitative huFcRI expression was matched for cells expressing the wild type (WT) and the cytoplasmic domain deletion mutant (MUT) by fluorescence activated cell sorting using anti-FcRI mAb 22.2-FITC (Medarex).

A polyclonal anti--chain Ab (666) was kindly provided by Dr. Jean-Pierre Kinet (21). In addition, polyclonal anti--chain Abs were prepared in rabbits immunized with a C-terminal peptide sequence that is shared by both human and murine -chain exactly as described (21). To verify the specificity of the polyclonal antibodies, -chain from U937 cells was immunoprecipitated with protein G-agarose bound anti--chain mAb (mAb 4D8) (kindly provided by Dr. J. Kochan) (22) followed by immunoblotting with the polyclonal Abs (see below).

Immunoprecipitation and Phosphotyrosine Analysis-- FcRI was immunoprecipitated from the transfected lines using either mAb 22.2 or mAb 197 (kindly provided by Dr. Paul Guyre, Dartmouth University Medical School) (23) pre-bound to protein G-agarose (Amersham Pharmacia Biotech, Piscataway, NJ). -Chain from transfected cells was immunoprecipitated by polyclonal rabbit anti--chain Abs bound to protein G-agarose (or from U937 cells with protein G-bound mAb 4D8). Cells (10-20 × 10⁶/ml) were lysed in PBS containing either 1% Nonidet P-40 (Sigma) or 1% digitonin (Wako Biochemicals, Waco, TX) and inhibitors (EDTA/pepstatin/aprotinin/sodium orthovanadate/pefabloc). Immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting.

For immunoblotting analysis, immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes (24). Membranes were blocked with 10% non-fat milk followed by incubation with either polyclonal anti--chain Ab or anti-phosphotyrosine mAb 4G10 (UBI). Blots were washed 3 times with PBS, 0.1% Tween 20 and bound mAb or Ab was detected with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Amersham Pharmacia Biotech or Jackson ImmunoResearch). Following 3 more washes, bound Ab was detected using ECL (Amersham Pharmacia Biotech) according to the manufacturer's directions. Membranes were stripped by incubation with Tris-HCl, pH 2.3, for 30 min at room temperature and then re-probed as described above.

Analysis of [Ca²⁺]_i-- Fura-2 (Molecular Probes, Eugene, OR), 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 (25). P388D1 cells, adhered to 25-mm diameter round glass coverslips at 5 × 10⁵ cells/ml, were incubated at 37 °C for 15 min with 2 µM fura-2 AM. During the last 5 min, anti-FcRIa mAb 22.2 F(ab')₂ was added. After incubation, the cells were washed once with modified PBS (PBS prepared with 5 mM KCl and 5 mM glucose) and then re-warmed to 37 °C for 5 min in modified PBS plus 1.1 mM Ca²⁺ and 1.6 mM Mg²⁺ prior to analysis. The coverslips were transferred to the stage of a Nikon Diaphot and the ratio of fluorescence emission of fura-2 was monitored. After establishment of a baseline, F(ab')₂ goat anti-mouse IgG was added at a final concentration of 35 µg/ml. Analysis was continued for an additional 5 min. Quantitation of intracellular [Ca²⁺] before and after treatment of cells with BAPTA-AM was performed using Indo-1 (Molecular Probes) in an SLM Spectrofluorometer (Spectronics Instruments, Rochester, NY) exactly as we have previously described (20, 25).

Endocytosis and Phagocytosis-- Endocytosis of transfected huFcRIa was determined by monitoring the disappearance of cell surface-associated anti-FcRI mAb 32.2 F(ab')₂ (Medarex) upon cross-linking with F(ab')₂ GM (26). Similarly, endocytosis of murine FcRIa on non-transfected cells was determined using mIgG2a (Sigma) and F(ab')₂ GM. Cells (50 µl, 5 × 10⁶/ml) were incubated with a saturating concentration of mAb for 15 min at 4 °C. Following two washes in PBS, 1% bovine serum albumin, F(ab')₂ GM was added, and cells were incubated for an additional 15 min at 4 °C. Cells were then placed at 37 °C for varying periods of time, rapidly pelleted, and washed with PBS, 1% bovine serum albumin containing azide at 4 °C. Remaining cell surface-associated receptor was quantitated with FITC-conjugated F(ab')₂ donkey anti-goat IgG by flow cytometry.

Phagocytosis by transfected P388D1 cells was determined in an adherent assay system (20). Biotinylated mAb 22.2 F(ab')₂ and biotinylated bovine erythrocytes were prepared as we have previously described (20). Biotinylated erythrocytes were saturated with streptavidin and washed. The resulting erythrocytes were coated with biotinylated mAb and the level of mAb binding was verified by flow cytometry.

P388D1 cells, adhered to round glass coverslips at 5 × 10⁵ cells/ml, were incubated with anti-FcRIa mAb 22.2 F(ab')₂-coated erythrocytes (E-22.2) in RPMI, 20% fetal calf serum (50 µl at 5 × 10⁷ E/ml) for 1 h at 37 °C. Alternatively, erythrocytes coated with an IgM anti-H2D^d (Pharmingen) were used. Non-internalized erythrocytes were lysed by brief immersion of the coverslip in dH₂O followed by immersion in buffer. Phagocytosis was quantitated by light microscopy and expressed as a phagocytic index (number of erythrocytes internalized per 100 P388D1 cells).

Treatment of cells with BAPTA-AM (Molecular Probes) to quench intracellular Ca²⁺ levels was performed as described previously (20). Briefly, coverslip adherent cells were incubated with varying concentrations of BAPTA-AM in RPMI, 20% fetal calf serum for 30 min at 37 °C followed by two washes. E-22.2 in RPMI, 20% fetal calf serum were then added and handled as described above. Controls included loading cells with the BAPTA-AM solvent (1% dimethyl sulfoxide) for the same period of time.

The kinetics of transfected FcRIa-specific phagocytosis was performed using a flow cytometric based assay (27). In this assay, the E-22.2 were labeled with the PKH26 Red Fluorescence Cell linker Kit (Sigma). Transfected P388D1 cells were mixed in suspension with labeled E-22.2 at a ratio of 50:1 (E:P388D1)(both in RPMI, 20% fetal calf serum), pelleted, and incubated at 37 °C for varying periods of time. At each time point, the supernatant was removed and non-internalized erythrocytes were rapidly lysed in hypotonic saline for 30 s followed by 3 washes in PBS, 1% bovine serum albumin at room temperature. Samples were analyzed immediately by flow cytometry. Results are expressed as a phagocytic capacity (mean fluorescence intensity of phagocytic cells with one or more internalized erythrocytes × % of cells with one or more internalized erythrocytes) as we have described (27).

Cytokine Analysis-- Cells were stimulated in 96-well tissue culture plates (Corning) with phorbol 12-myristate 13-acetate, surface absorbed rabbit IgG, or surface absorbed F(ab')₂ GM IgG + mAb 22.2 F(ab')₂. Wells were coated with absorbed protein (20 µg/ml rabbit IgG or F(ab')₂ GM) for 2 h at 37 °C. For anti-FcRI mAb 22.2 F(ab')₂ stimulation, mAb at 20 µg/ml was added to F(ab')₂ GM-coated wells for 1 h at 37 °C. Cells (1-2.5 × 10⁵ cells/ml) were added to the wells and cultured for varying periods of time. Levels of murine cytokines in diluted culture supernatants were quantitated by enzyme-linked immunosorbent assay. For IL-1 determination, recombinant standard, capture ab (polyclonal rabbit Ab), and biotinylated detection and neutralization mAb (clone 1400.24.17) were obtained from Endogen (Woburn, MA). For IL-6 determination, recombinant standard, capture mAb (clone MP5-2-F3), and biotinylated detection mAb (clone MP5-32C11) were obtained from Pharmingen. Horseradish peroxidase-conjugated streptavidin (Jackson) and then TMB substrate were added and the A_450 nm was determined.

Flow Cytometry-- Aliquots of cells at 5 × 10⁶ cell/ml were incubated with saturating concentrations of primary mAb for 30 min at 4 °C followed by two washes. For indirect immunofluorescence, the cells were then incubated with saturating concentrations of FITC-conjugated goat anti-mouse IgG F(ab')₂ at 4 °C for another 30 min. After washing, the cells were analyzed immediately for immunofluorescence using a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA).

Statistical Analysis-- Analysis of flow cytometry listmode data was done using CellQuest (Becton Dickinson Immunocytometry). Statistical comparisons were performed with the paired t test. A probability of 0.05 was used to reject the null hypothesis that there is no difference between the samples.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Assembly of FcRIa Receptor Complexes-- To investigate the functional significance of the cytoplasmic domain of human FcRIa, P388D1 cells stably transfected with cDNA encoding the full-length wild type FcRIa (WT) or a cDNA encoding a cytoplasmic domain deletion mutant form of FcRIa (MUT) were studied. Transfected cell lines were sorted to generate clones with identical levels of receptor expression (Fig. 1) that were used in all subsequent studies.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Expression of human WT and MUT FcRIa on the surface of stably transfected P388D1 cells. Cells were incubated with a saturating concentration of the anti-human FcRIa mAb 22.2-FITC and analyzed by flow cytometry.

Human FcRIa expressed on monocytes and the myelomonocytic cell line U937 noncovalently associates with the -chain of the FcRI receptor complex (9, 10). The transmembrane regions of FcRIa and the -chain mediate receptor complex assembly and 20 of the 21 amino acids in the transmembrane region are identical in murine and human -chain with one conservative difference (I  V). As predicted, comparable amounts of -chain was co-immunoprecipitated from the WT and MUT lines after cell lysis in buffer containing 1% digitonin (Fig. 2). As a positive control, we co-immunoprecipitated -chain with endogenously expressed murine FcRII and FcRIIIa using mAb 2.4G2. In accordance with the -chain association data, cross-linking of the transfected WT and MUT huFcRIa with mAb 22.2 F(ab')₂ + F(ab')₂ GM resulted in tyrosine phosphorylation of the -chain (results not shown) demonstrating that both WT and MUT huFcRIa associate with the endogenous murine -chain and initiate tyrosine phosphorylation of the -chain.

View larger version (73K): [in this window] [in a new window]   Fig. 2.   Co-immunoprecipitation of murine -chain with WT and MUT human FcRIa. Cells were lysed in buffer containing 1% digitonin as described under "Experimental Procedures." Human FcRIa was immunoprecipitated with the anti-FcRIa mAb 22.2 bound to protein G-agarose. Murine FcRII/FcRIII was immunoprecipitated with the anti-FcRII/III mAb 2.4G2 bound to protein G-agarose. -Chain was detected with a polyclonal anti-serum (number 8224) prepared against a C'-terminal peptide as described under "Experimental Procedures."

The huFcRIa CY Domain Alters the Magnitude and Kinetics of FcRIa Internalization-- While devoid of tyrosine residues, the -chain of murine FcRIa has been shown to be phosphorylated on serine and/or threonine residues (17) and human FcRIa has been shown to bind to ABP under some conditions (18). Furthermore, FcRIa in the absence of the -chain can signal for calcium in COS-1 cells and the transmission of this calcium signal requires the FcRIa cytoplasmic domain (19). Accordingly, we considered the possibility that the CY domain of huFcRIa may contribute to the functional properties of the receptor complex. Using erythrocytes coated with the anti-human FcRIa mAb 22.2 F(ab')₂, both WT and MUT huFcRIa mediated receptor-specific phagocytosis (Fig. 3). However, the WT construct consistently displayed a higher phagocytic index despite identical levels of receptor expression (Fig. 3). There was no internalization of E-22.2 by parental non-transfected P388D1 cells and no phagocytosis of erythrocytes coated with an IgM anti-H-2Dd mAb (clone 3-25.4) by any cell type (Fig. 3A), despite comparable binding of the E-3-25.4 probe to the transfected cells when compared with E-22.2.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Receptor-specific phagocytosis by WT and MUT human FcRIa. A, quantitation of human FcRIa-specific phagocytosis (E-22.2 F(ab')2, hatched bars, n = 15) or H-2Dd-specific phagocytosis (E-3-25.4, solid bars, n = 3) by WT and MUT human FcRIa stable transfectants. As a control, parental non-transfected cells were analyzed. Phagocytosis was performed as described under "Experimental Procedures" and quantitated by light microscopy. Data are expressed as the mean phagocytic index ± S.D. B, kinetics of phagocytosis of E-22 F(ab')2 by WT () and MUT () human FcRIa P388D1 stable transfectants. Phagocytosis was performed as described under "Experimental Procedures" and quantitated by flow cytometry. Data are presented from a single representative experiment (n = 5). *, p < 0.001, FcRIa-specific phagocytosis by MUT versus WT.

We considered the possibility that engagement of the ligand-binding site of FcRIa, which can augment receptor function (28), might alter the difference in phagocytic capacity between the WT and MUT FcRI. However, saturation of FcRIa with mIgG2a did not alter the magnitude of FcRIa-specific phagocytosis and did not abrogate the quantitative difference in phagocytosis between WT and MUT FcRIa (Table I).

View this table: [in this window] [in a new window]   Table I FcRIa-specific phagocytosis Coverslip adherent cells were prepared as described under "Experimental Procedures." Murine IgG2a (20 µg/ml) was added to appropriate coverslips, incubated for 10 min at room temperature followed by the addition of E-22.2 F(ab')2 to all of the coverslips. After 60 min at 37 °C, non-internalized E were lysed and the phagocytic index (the number of internalized erythrocytes per 100 P388D1 cells) was determined by light microscopy.

Phagocytosis by WT huFcRIa also displayed more rapid kinetics (Fig. 3B). Similarly, while both WT and MUT huFcRIa were capable of endocytosis, endocytosis by the WT receptor was more rapid than that mediated by the MUT receptor (Fig. 4). Endocytosis of endogenous murine FcRIa, assessed on non-transfected P388D1 cells, was indistinguishable from the transfected huFcRIa. Since we have matched the WT and MUT cell lines for receptor expression, the differences in phagocytic capacity and the more rapid kinetics of phagocytosis and endocytosis by WT FcRI provide the first evidence that the cytoplasmic domain of FcRIa, in association with the -chain, can affect receptor function.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Receptor-specific endocytosis by WT and MUT human FcRIa in P388D1 stable transfectants and of murine FcRI in non-transfected P388D1 cells. Internalization of WT huFcRIa (), MUT human huFcRIa (), and murine FcRI () after receptor-specific cross-linking was determined by flow cytometry as described under "Experimental Procedures." Data are presented as the mean ± S.D. from a total of eight experiments. *, p < 0.01 (1 min) and p < 0.005 (2 min and 5 min), WT versus MUT % internalized.

The CY Domain of the -Chain Determines Ca²⁺ Sensitivity of FcRIa Phagocytosis-- Through the use of chimeric and mutant receptors, the ITAM has been shown to be both necessary and sufficient for FcR phagocytosis and the FcR Ca²⁺ transient (14, 15, 20, 29-31). The functional importance of the Ca²⁺ transient has been demonstrated with huFcRIIa which incorporates an ITAM directly in the CY domain and requires elevations in intracellular Ca²⁺ to mediate phagocytosis (20). In contrast, FcRIa/-chain specific phagocytosis is independent of the receptor-induced Ca²⁺ transient (20), and we considered the possibility that the CY domain of FcRIa confers a Ca²⁺-independent phenotype on FcRIa-specific phagocytosis. When intracellular Ca²⁺ levels were quenched with BAPTA (resulting in [Ca²⁺] = 57 ± 9.3 nM with 20 µM BAPTA treatment), receptor-specific phagocytosis induced by the WT huFcRIa was unaltered (Fig. 5A), as we have previously shown for FcRI on human monocytes. In contrast, receptor-specific phagocytosis induced by the MUT huFcRIa was blocked by pretreatment of the cells with BAPTA in a dose-dependent manner (Fig. 5A).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Differential sensitivity to pretreatment with BAPTA. A, cells were pre-loaded with the intracellular Ca2+ chelator BAPTA followed by the addition of E-22.2 F(ab')2 to assess human FcRIa-specific phagocytosis in WT and MUT P388D1 stable transfectants (n = 8). WT (open bars) and MUT (hatched bars) P388D1 stable transfectants were incubated with E-22.2 were prepared at maximal mAb conjugation ratios (prepared as described in the legend to Fig. 3A). Alternatively, WT P388D1 stable transfectants (solid bars) were incubated with E-22.2 prepared at lower conjugation ratio to match the quantitative level of phagocytosis of the MUT huFcRIa (see text). B, as a control, FcRIIa-specific phagocytosis in P388D1 cells transfected with human FcRIIa (20) was determined using E-IV.3 Fab (n = 6). Data are expressed as the mean ± S.D. *, p < 0.01 relative to control (no BAPTA). N.D., not done.

Since the absolute level of MUT huFcRIa phagocytosis is lower than WT huFcRIa, we considered the possibility that the BAPTA sensitivity might be related to the quantitative level of phagocytosis. Accordingly, phagocytosis by the WT huFcRIa was performed with E-22.2 prepared with a lower mAb conjugation level resulting in a phagocytic index of 65.1 ± 13.2 compared with a phagocytic index of 70.4 ± 6.2 for MUT huFcRIa and E-22.2 prepared at the maximal conjugation level. WT huFcRI insensitivity to BAPTA was maintained under these reduced phagocytic conditions (Fig. 5A) indicating that the Ca²⁺ insensitivity is a property of the CY domain of huFcRIa. As an additional control, FcRIIa-specific phagocytosis by P388D1 cells expressing full-length huFcRIIa (with a phagocytic index of 167 ± 32.6) was also shown to be blocked by pretreatment of the cells with BAPTA (Fig. 5B), as we have previously reported (20). Importantly, both WT and MUT huFcRIa receptor complexes induced indistinguishable Ca²⁺ transients when cross-linked with anti-receptor mAb (results not shown). Thus, WT huFcRIa engages a Ca²⁺-insensitive phagocytic pathway while MUT huFcRIa with the associated -chain engages a Ca²⁺-sensitive phagocytic pathway. These results provide additional evidence that the CY domain of the ligand-binding -chain of huFcRIa alters functional properties of -chain ITAM-dependent functions.

Requirement of the FcRIa -Chain for the Induction of IL-6 Secretion-- In addition to its role in internalization, FcRIa can also modulate the immune response through the induction of cytokine secretion. In particular, activation of monocytes/macrophages by FcRIa can result in the secretion of IL-6 and IL-1 (32, 33). Accordingly, P388D1 expressing the WT and MUT forms of huFcRIa were stimulated with receptor-specific mAb bound to surface absorbed F(ab')₂-GM. Quantitation of IL-1 secretion after cross-linking of huFcRIa demonstrated that both WT and MUT forms of the receptor were capable of eliciting comparable levels of secretion of this cytokine (Fig. 6). Cells incubated in the presence of GM alone were not stimulated to secrete IL-1 above the baseline control. In contrast, cross-linking WT huFcRIa, but not MUT FcRIa, induced the secretion of IL-6 (8-h time point). We did detect IL-6 production above baseline at 24 h after MUT FcRIa stimulation; however, neutralization of endogenously produced IL-1 prevented this induction of IL-6 secretion by the MUT FcRIa after 24 h culture (253 ± 52 pg/ml and 75 ± 21 pg/ml in the absence and presence of a neutralizing anti-IL-1 mAb). In contrast, neutralizing anti-IL-1 mAb did not abrogate the IL-6 induction observed at the 4- or 8-h time points by WT huFcRIa. No significant difference in the ability of phorbol 12-myristate 13-acetate (100 ng/ml) or surface bound IgG, engaging endogenous murine FcRIIa/FcRIIIa and transfected human FcRIa, to elicit IL-1 or IL-6 secretion was observed between the WT and MUT lines (results not shown). These results document the requirement for the -chain of the FcRIa receptor complex for the induction of the IL-6 response by the receptor complex and demonstrate that the pathways leading to IL-6 secretion and IL-1 secretion are distinct.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   IL-6 release, but not IL-1 release, requires the cytoplasmic domain of FcRIa. P388D1 stable transfectants were cultured for 8 h in tissue culture wells that had been pretreated with F(ab')2 GM (XL) or mAb 22.2 F(ab')2 + XL as described under "Experimental Procedures." Data are expressed as the mean pg/ml cytokine produced ± S.D. (n = 6). *, p < 0.01 relative to the XL alone control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Three lines of evidence indicate that the FcRIa·-chain complex induced responses are altered by the cytoplasmic domain of the -chain. First, when matched for receptor expression, WT FcRI has a higher phagocytic capacity than the cytoplasmic domain deletion mutant. The WT receptor complex also displays faster kinetics of phagocytosis and endocytosis despite comparable kinetics of tyrosine phosphorylation of the -chain. Second, the cytoplasmic domain of FcRI confers a calcium-independent phenotype on phagocytosis by the receptor complex. Third, and most importantly, we have observed that the induction of IL-6 secretion requires the cytoplasmic domain of the FcRIa -chain (Fig. 6). Taken together, these data provide the first direct demonstration of a functional role of the cytoplasmic domain of FcRI in the FcRIa·-chain complex.

Previous studies of FcRI transiently transfected into COS cells have suggested that cross-linking of the -chain may mediate changes in intracellular calcium and endocytosis (19, 34, 35). However, in the absence of -chain, FcRIa is not competent for phagocytosis (13-15). The central role of the -chain and the tyrosine kinase p72^Syk, which docks via SH2 domains to the tyrosine-phosphorylated -chain, has been demonstrated through a series of experiments using co-transfection of the FcRIa·-chain complex with Syk kinase and transfection of chimeric receptor constructs incorporating either the -chain cytoplasmic domain or Syk kinase directly (36, 37). Furthermore, the observation that both 1) human monocytes/macrophages in which Syk has been specifically ablated by antisense constructs (38) and 2) murine macrophages from Syk knockout mice (39, 40) are incapable of Fc receptor-mediated phagocytosis supports the conclusion that the -chain induced activation of Syk is necessary and sufficient for a phagocytic response. Given these results, our observations that the cytoplasmic domain of FcRIa modulates the kinetics of phagocytosis may reflect the association of cytoskeletal elements with the cytoplasmic domain and subsequent changes in the mobility of the receptor in the plasma membrane. Such a mechanism may explain the observation that FcRI in the NOD mouse also shows altered kinetics of endocytosis (41).

The change in calcium independent signaling elements and in the induction of IL-6 synthesis indicates that the cytoplasmic domain of FcRI also affects the nature of intracellular signals generated by the FcRI·-chain receptor complex. Studies of the high affinity receptor complex for IgE comprised of ligand binding -chain, a single -chain, and a -chain homodimer indicate that the -chain is constitutively associated with the Src kinase Lyn and can recruit protein kinase C- both of which can modulate the signaling capacity of the ITAM in the -chain (42-45). In this manner, the -chain acts as an amplifying mechanism and, although the role of serine/threonine phosphorylation of -chain is not yet established, the presence of serine/threonine phosphorylation targets within the -chain provide an attractive target for regulation. Indeed, FcRIa cross-linking on U937 cells results in the serine phosphorylation of the -chain (46) and recruitment of protein kinase C- and protein kinase C- to the membrane by Fc receptors during phagocytosis has been documented (47). In a parallel fashion, perhaps, the cytoplasmic domain of FcRIa may recruit signaling elements to the receptor complex. Although it contains no ITAM the FcRI cytoplasmic domain is actively serine/threonine phosphorylated and dephosphorylated (17), suggesting active participation in receptor function. Thus, through regulation of phosphorylation and/or recruitment of other signaling molecules to the receptor complex, it is also possible that the FcRIa cytoplasmic domain may directly transmit the signal for IL-6 release in the absence of participation of the -chain.

It is also interesting to note that subtle differences in the ITAM sequences used by Fc receptors may also contribute to distinct biological properties. The ITAM-like sequence in FcRIIa differs from the ITAM in the -chain and this difference, or other adjacent sequences, influence the relative dependence on intracellular calcium transients for phagocytosis (Fig. 5). This property and the differences in serine/threonine residues within the ITAM may allow for differences in some of the functions between FcRIIa and FcRIa/FcRIIIa.

The mechanism(s) by which the cytoplasmic domain of FcRIa alters receptor function is not clear. Murine FcRI is phosphorylated on serine/threonine residues after phorbol 12-myristate 13-acetate stimulation (17). However, the functional importance of this phosphorylation has not been examined. Differences in the association of WT and MUT FcRIa with -chain cannot be the mechanism for the observed functional differences between these receptors. Transfectants have been sorted for identical cell surface receptor expression and both WT and MUT associate equally with -chain (by direct co-immunoprecipitation and functionally by receptor induced tyrosine phosphorylation). Recent studies have also demonstrated that association with -chain is essential for stable expression of huFcRIa (11, 12). A direct interaction between human FcRIa and ABP-280 (non-muscle filamin) has been reported (18). Although there are no know consequences of the interaction between FcRIa and ABP, ligand engagement of FcRIa results in both the dissociation of the receptor from ABP-280 and in the enhancement of the FcRIa triggered oxidative burst in U937 cells (28). In the present study, we have used an anti-human FcRIa mAb F(ab')₂ fragment that does not engage the ligand-binding site (23). In this transfection system, saturation of FcRI with mIgG2a did not alter the magnitude of human FcRIa-specific phagocytosis nor did it abrogate the quantitative difference in phagocytosis between WT and MUT FcRIa (Table I).

Demonstration that the cytoplasmic domain of the -chain of FcRIa alters the functional properties of the FcRI·-chain complex provides a mechanism for unique biologic properties initiated by each receptor complex. Given the diversity in primary sequence of the cytoplasmic domains of the /-chain-associated Fc receptors, the opportunity for these unique domains to confer distinct functions on each receptor complex is clear. These observations also suggest that single nucleotide polymorphism leading to missence mutations in the cytoplasmic domains of these receptors may have biological significance. We have recently described two single nucleotide polymorphisms in the cytoplasmic domain of FcRIa proximate to putative phosphorylation sites (48). These polymorphisms may provide another level of functional variation which builds upon a general framework of the role of sequence variations in the cytoplasmic domains altering the functional properties of the receptor complex.

	ACKNOWLEDGEMENTS

We thank Ka Chen, Jessica T. Leonard, Patricia B. Redecha, Dana Lau, Paul Palavin, and James J. Moon for technical assistance and Andrew J. Beavis for flow cytometric analysis and cell sorting.

	FOOTNOTES

^* This work was supported by National Institutes of Health (NIH) Grants RO1-AR33062, RO1-AR42476, and AI-22193, flow cytometry was supported in part by NIH core Grants P60-AR38320 (Hospital for Special Surgery) and P60-AR20614 (University of Alabama at Birmingham).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.

^§ To whom correspondence and reprint requests should be addressed: Div. of Clinical Immunology and Rheumatology, Dept. of Medicine, 433A Tinsley Harrison Tower, 1900 University Blvd., Birmingham, AL 35294-0006. Tel.: 205-934-0894; Fax: 205-934-1564; E-mail: JEdberg@ uab.edu.

	ABBREVIATIONS

The abbreviations used are: IL, interleukin; ITAM, immunoreceptor tyrosine activation motif; mAb, monoclonal antibody; PBS, phosphate-buffered saline; BAPTA-AM, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester; FITC, fluorescein isothiocyanate; [Ca²⁺]_i, intracellular [Ca²⁺].

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES