©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Role for IgG-Fc
TRANSDUCTIONAL POTENTIATION FOR HUMAN HIGH AFFINITY Fc RECEPTOR (FcRI) SIGNALING (*)

(Received for publication, December 8, 1994)

Lorraine C. Pfefferkorn (1)(§) Jan G. J. van de Winkel (2) Sharon L. Swink (1)

From the  (1)Department of Microbiology, Dartmouth Medical School, Lebanon, New Hampshire 03756 and the (2)Department of Experimental Immunology, University Hospital Utrecht, Utrecht, The Netherlands

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human type 1 Fc receptors (FcRI) bind with high affinity (K = 10M) Fc regions of monomeric IgG1 and IgG3. As demonstrated in this report, interaction of IgG-Fc with the ligand binding site on FcRI alters its capacity for aggregation-dependent signaling. This Fc-dependence was demonstrated in normal monocytes and U937-10.6 cells exposed to monomeric IgG and then to anti-FcRI F(ab`)(2) that cross-link the receptor. Using O(2) production to measure cell signaling, we found that binding by high affinity IgGs of various species, as well as by murine hybrid IgGs containing only one high affinity heavy chain, resulted in a marked increase in FcRI-mediated signaling. Preaggregated FcRI/IgG had a ratio of one. IgG binding after aggregation of unligated FcRI did not restore signaling. Dose responses indicated that concentrations of IgG that saturated FcRI optimized transductional activity. The inclusion of unligated with ligated FcRI in aggregates depressed activity, indicating a lack of trans-activation of unligated FcRI. Significantly, IgG-binding markedly increased aggregation-dependent tyrosine phosphorylation of FcRI -chains and the association of tyrosine phosphorylated Syk. Thus, the consequences of IgG-Fc binding were increases in aggregation-dependent phosphorylation of FcRI -chains, recruitment of pp72Syk to FcRI, and signaling of the NADPH oxidase pathway.


INTRODUCTION

Fc receptors (FcR) (^1)bind the Fc region of immunoglobulin G (IgG) and are expressed on the surface of a variety of cells. The human high affinity FcR (FcRI) is a multichain receptor comprised of a 70-kDa glycoprotein alpha-chain and homodimeric largely intracellular -chain subunits(1, 2) . Surface expression of the receptor on monocytes and U937 cells is induced by IFN-(3, 4, 5) . FcRI binds with high affinity (K= 3 nM) human IgG subclasses IgG1 and IgG3. It has one log less affinity for IgG4 and no apparent affinity for IgG2(6, 7) . The receptor binds with high affinity certain IgGs from other species that possess sequence homologies in the C2 Fc regions(6) . FcRI engagement of complexed IgG or anti-receptor antibodies(8, 9) aggregates the receptor(10, 11) , triggering several very early responses including fluxes of intracellular Ca(12, 13) , tyrosine phosphorylations of several proteins (14) and activation of the NADPH oxidase pathway(12) .

Myelocytic signaling functions of FcRI, FcRIIIA, FcRI, and FcalphaR (15) are mediated by tyrosine activation of -chains (10, 11, 13) in motifs recognized by members of src family(16) . Src kinases Hck and Lyn associate with FcRI -chains to constitute a preaggregation multichain transduction unit(17) . Aggregation of FcRI induces the tyrosine phosphorylation of its -chains(15, 18) and the tyrosine phosphorylation of Syk kinase(18, 19, 20) . Syk, a 72-kDa structural homologue of the T-cell signal-transducing ZAP70, is recruited to -chains on activated FcRI(21) .

We have previously reported that an equimolar mixture of anti-FcRI monoclonal antibodies 32 and 22 is a potent combination for triggering FcRI-mediated NADPH oxidase activity(8, 9) . Subsequently, we found that the F(ab`)(2) fragments of these anti-FcRI trigger poorly, and that this situation is reversed by binding high affinity IgG to the receptors(22) . For this report, we analyzed the consequences of IgG-binding on signaling through FcRI. By engaging the ligand binding site on FcRI with monomer ligand and then cross-linking the receptors through anti-FcRI F(ab`)(2), we show evidence of a novel dependence on IgG-Fc for FcRI transductional activity. We show that IgG-Fc binding increased aggregation-dependent O(2) production, tyrosine phosphorylation of FcRI -chains, and the association of pp72Syk kinase to activated FcRI.


MATERIALS AND METHODS

Cells

U937 cells (10.6 subclone) were cultured for 5-6 days in RPMI 1640 medium containing 10% fetal bovine serum, gentomycin, and 20 units/ml IFN- (Genentech, S. San Francisco, CA) (23) . Monocytes were obtained by cytopheresis and purified on Ficoll-Hypaque as described(24) ; further purification was through repeated rate-sedimentation separations from lymphocytes until >90% purity was achieved. Monocytes were cultured for 2 days in the RPMI medium containing 20 units/ml IFN-.

Antibodies and Immunoglobulins

Purified anti-FcRI mAbs 32 and 22, 32 F(ab`)(2) and 22 F(ab`)(2), and anti-FcRII IV.3 Fab were obtained from Dr. R. Graziano at Medarex, Annendale, NJ. Affinity-purified sheep F(ab`)(2) anti-murine IgG-F(ab`)(2) and goat F(ab`)(2) anti-murine IgG were obtained from Organon Technika (Durham, NC). Covalently cross-linked conjugates of 22Fab with high unit multiplicity were prepared (25) and provided by Dr. M. W. Fanger (Dartmouth Medical School, Lebanon, NH). Monomer Igs in this paper include human IgG1 (hIgG1) purified from myeloma serum as described previously(8) ; purified human serum IgG subclasses IgG1, IgG2, IgG3, and IgG4 (The Binding Site LTD, (Birmingham, UK); purified goat and rabbit IgG (Organon Technika); hybrid murine IgGs (anti-human IgA1 (2a/2a), anti-human IgA/anti horseradish peroxidase (2a/2b), and anti-horseradish peroxidase (1/2a) were prepared by Dr. J. G. J. van de Winkel as described(26) ); and murine mAbs of the IgG1, IgG2a, and IgG2b subclasses, murine mAbs of the IgM, IgA, and IgE classes, and rat mAbs of the IgG1, IgG2a, and IgG2b subclasses. mAbs used as ligands were obtained from hybridoma cultures. mAbs are identified by their hybridoma ATCC designations or mAb names in the order of the Fig. 2B list: m2a-1, -2, -3 are murine IgG2a mAbs w632, HB63, HB32; m2a+2b represents murine IgG2a plus mAb IgG2b BBMI; r2b-1,-2 are rat IgG2b mAbs TIB128, TIB129; m2b-1, -2, -3 are murine IgG2b mAbs OKMI, IV.3, AML 2-23; m1-1, -2, -3, -4, -5, -6, -7, -8 are murine IgG1 mAbs 3G8, P3, TIB191, TIB139, HB205, HB195, HB202, HB203; r1-1 is rat IgG1 mAb TIB168; r2a-1 is a rat IgG2a mAb TIB166; mM-1, -2, -3 are murine IgM mAbs PM81, PMN6, TIB200; mA-1 is murine IgA mAb TIB196; mE-1 is murine IgE mAb TIB141.


Figure 2: Effects of Igs of various species, class, and subclass on FcRI signaling. Cells were preincubated in various Igs and, after a brief wash, were analyzed for the percent occupancy of FcRI-ligand binding sites and for FcRI-mediated O(2) production. A, preincubations contained hIgG1 (H), goat IgG (G), rabbit IgG (R) (10 µg/ml), or medium (none). B, preincubation media were hybridoma supernatants (200 µl) containing murine (m) or rat (r) mAbs of IgG subclasses 2a (2a), 1 (1), and 2b (2b) or of the IgM (M), A (A), or E (E) classes. Identities of mAbs are listed under ``Materials and Methods''; those with reactivity toward monocyte surface antigens are indicated (*). Assays of mAb-preincubated cells included control cells preincubated with hIgG1 or medium. In B, the O(2) production by cells receiving 32F(ab`)(2) and 22F(ab`)(2) but no IgG was subtracted. Therefore, data in B represent the enhancement of O(2) production due to ligand. (Negative values are slight depressions in response by unligated receptors.)



Assay for O(2) Production

O(2) production was measured by luminol-enhanced chemiluminescence(27) . Cells were incubated at 22 °C for 20 min with primary antibody or ligands (10 µg/ml or in hybridoma supernatants) after which they were suspended in O(2) assay medium (15) at a concentration of 2-5 times 10^6/ml. Cells were stimulated by mixing with an equal volume of 10M luminol (5-amino-2,3-dihydrophthalazine-1,4-dione) containing anti-FcRI F(ab`)(2), 22Fab times 22Fab conjugates, or polyclonal anti-murine IgG antibodies as described previously(15) . Luminometry on a BioOrbit luminometer 1250 was used to measure the instantaneous velocity of O(2) production at 25 °C; data was recorded in mV/s. Measurements were recorded every 0.5-1.5 min until the respiratory burst subsided to 10% of peak activity (about 10-15 min). For relative totals of O(2) in bursts, velocity data were integrated and are expressed in mV. Chemiluminescence of cells lacking stimulants was subtracted.

FACS Analysis of Cell-associated Antibodies and Ligands

Cells were incubated at 22 °C for 20 min with primary antibody or ligand (10 µg/ml or in hybridoma supernatants). After washing 3 times with PBS containing 1 mg/ml BSA (PBS/BSA), cell samples were incubated at 4 °C for 90 min with PBS/BSA containing one of the following FITC-conjugated second antibodies or FITC-hIgG1: FITC-conjugated F(ab`)(2) goat anti-murine IgG (FITC-GAM) (CALTAG, San Francisco, CA), FITC-conjugated hIgG1 (FITC-hIgG1) prepared by conjugating purified hIgG1 with fluoroisothiocyanate, and FITC-conjugated rat IgG2a mAb against the murine Ig kappa-chain (FITC-anti-kappa) (Zymed, San Francisco, CA). After washing 3 times, the cells were fixed in 1% paraformaldehyde in PBS. Cell-associated fluorescence was analyzed using a FACScan flow cytometer (Becton Dickinson)(9) ; measurements of the mean fluorescence intensity/cell were made on a 4-log scale. Data were converted to FITC/cell using a standard curve generated from microspheres of known FITC content (Flow Cytometry Research Corporation, San Juan, Puerto Rico) as described previously(15) . For assays using FITC-anti-IgG, data are expressed as FITC-second antibody binding sites/cell. For assays using FITC-hIgG1, cells were exposed to various unlabeled IgGs, and their binding was determined by the extent to which subsequent FITC-hIgG1 (10 µg/ml) was blocked. Thus, 100% binding by IgG means 100% blocking of FITC-hIgG1 after subtraction of nonspecifically associated FITC-hIgG1 (which was measured in the presence of 10 mg/ml hIgG). For assays using FITC-anti kappa-chain mAb, calculations for the number of kappa-chains/cell were attained by correcting FITC/cell values for the FITC/second antibody ratio.

Aggregation and Internalization

FcRI aggregation was assessed by measuring cross-linking-induced receptor internalization. Internalization was measured by fluorescent labeling and FACS analysis. Cells were preincubated for 20 min at 22 °C in O(2) assay medium containing 32F(ab`)(2) with or without ligand. FcRI were cross-linked by adding 5 µg/ml FITC-22F(ab`)(2) and incubating at 37 °C for 25 min. Time course assays established that induced internalization did not continue after 25 min. The cells were washed with cold PBS/BSA and incubated again at 4 °C for 60 min with PBS/BSA (100 µl) or PBS/BSA containing 100 µg/ml rabbit anti-FITC antibodies (Molecular Probes, Eugene, OR) to block fluorescence of cell surface FITC. To assess the efficiency of this blocking, the procedure using 32F(ab`)(2) and ligand binding was the same, but exposure to FITC-22F(ab`)(2) was done at 4 °C for 60 min. Subsequent washes and incubation with and without anti-FITC Abs was the same as before. After a final wash, fluorescence was measured by FACScan analysis and data converted to FITC second antibody binding sites/cell. Internalization was calculated according to the equation A - ((T - A)F) = I, where T = total sites/cell; A = total sites/cell of anti-FITC blocked cells; (T - A) = surface sites blocked by anti-FITC; F = correction factor for percent blocking efficiency of anti-FITC, obtained from the percent unblocked/percent blocked fluorescence of the 4 °C control procedure; I = internalized sites/cell.

Immunoprecipitation, Immunoblotting, and Autoradiography

Cells (2 times 10^7/ml) were preincubated for 20 min at 22 °C in O(2) assay medium with or without hIgG1 (10 µg/ml) and then for 5 min at 37 °C after the addition of an equal volume of luminol containing 22F(ab`)(2) and/or 32F(ab`)(2) (5 µg/ml each). Cells were rapidly chilled and washed in cold PBS containing 200 uM Na(3)VO(4). They were solubilized for 30 s in 4 °C lysis buffer containing 0.5% Nonidet P-40 and 0.5% digitonin, and then for another 10 min after the addition of an equal volume of lysis buffer containing 1% digitonin. Both buffers contained aprotinin (0.23 units/ml), 2 mM phenylmethylsulfonyl fluoride, 5 mM Na(2)EDTA, 10 mM NaF, and 200 uM Na(3)VO(4) in PBS, pH 7.4. After a 10-min centrifugation at 16,000 times g, supernatants were agitated with goat anti-mouse Ig kappa-chain (Pierce, Rockford IL) conjugated to Biosupport beads AB1 according to instructions of the manufacturer. Beads were washed 3 times with lysis buffer, and bead-precipitated proteins were separated by nonreducing SDS-polyacrylamide gel electrophoresis on 16% acrylamide gels followed by transfer to nitrocellulose membranes. Membranes were blocked in 5% BSA, and immunoblotted with rabbit anti-phosphotyrosine antibody (anti-PY) (a gift of Gustav Leinhard, Dartmouth Medical School) or rabbit anti--chain antibody (anti-) (a gift of J.-P. Kinet, NIH, NIAID). For immunoblotting with rabbit anti-Syk antibody (UBI, Lake Placid, NY), membranes were blocked and blotted in 5% nonfat milk. Immunoblotting and membrane stripping were performed as described previously(15) . Horseradish peroxidase-conjugated anti-rabbit antibodies and the ECL system (Amersham, Arlington Heights, IL) were used to develop blots. Positions corresponding to nonreduced and phospho- dimers have been previously established(15) .


RESULTS

IgG-Fc Binding Increases FcRI-mediated Signaling

To determine whether signaling through FcRI is altered by IgG-binding to its ligand binding site, we preincubated 10.6 cells with or without hIgG1 and reacted the cells with anti-FcRI mAb 32 and 22(9, 28) F(ab`)(2) fragments. These anti-FcRI bind to nonoverlapping epitopes outside of the FcRI ligand binding site and trigger through receptor cross-linking(8, 9) . The effect on cell signaling was analyzed by measuring the production of O(2). As shown in Fig. 1A, cross-linking elicited a substantially greater respiratory burst from cells that had been preincubated with hIgG1. A second assay performed in the presence of FcRII-blocking IV.3Fab gave similar results, indicating that the effect IgG1 had on oxidase activity did not involve FcRII.


Figure 1: The effect of hIgG on FcRI-mediated O(2) production. A, U937 10.6 cells, differentiated by IFN- for 6 days, were preincubated for 10 min in O(2) assay medium with and without hIgG1 (G1) (10 µg/ml). IV.3Fab (25 µg/ml) was added to block FcRII. The preincubated cells were added to luminol containing 32F(ab`)(2) and 22F(ab`)(2) (F) (5 µg/ml each), or 22F(ab`)(2) alone as control. O(2) production was measured by luminometry and is expressed in mV ± S.D. Controls were subtracted. B, human monocytes cultured with IFN- for 2 days were preincubated with or without hIgG1 for 5 min and stimulated by the addition of cells to luminol containing 5 µg/ml intact mAbs 32 and 22 (W) or 32F(ab`)(2) and 22F(ab`)(2) (F). C, U937 10.6 cells were preincubated with human IgG subclasses 1, 2, 3, and 4, or no IgG (none) for 10 min and then washed. Duplicate cell samples were stimulated by the addition of 32F(ab`)(2) and 22F(ab`)(2) for an O(2) assay. Another set of duplicates were incubated with FITC-hIgG1 and analyzed by FACS to measure hIgG subclass binding. O(2) production and IgG-binding to FcR are expressed as a percent of the hIgG1 control. There was no detectable binding by hIgG2. Data represent the mean of three experiments.



A similar assay was performed with monocytes that were treated with IFN- to induce the surface expression of newly synthesized FcRI. Treatment was done in culture medium lacking IgG so that induced receptors would be occupied by high affinity ligands. When anti-FcRI F(ab`)(2) triggered receptors in the absence of IgG, there was almost no activity (Fig. 1B), but monocytes that had been preincubated with IgG produced a respiratory burst that was comparable with that triggered by intact anti-FcRI. We conclude that IgG binding profoundly affected FcRI signaling in normal human receptors.

To determine whether other human subclasses exert a similar effect, we preincubated 10.6 cells with hIgG subclasses 1, 2, 3, and 4; washed the cells briefly; and then cross-linked their FcRI. As shown in Fig. 1C, hIgG1- and hIgG3-preincubated cells had enhanced FcRI signaling, whereas hIgG4 preincubation produced an intermediate response. FACS analysis showed that bound IgG-binding to the cells (Fig. 1C) correlated with enhanced O(2) production (Fig. 1C). HIgG2 did not bind and had no effect on signaling over the IgG-free control.

Certain other nonhuman IgGs bind human FcRI. To analyze their effect specifically on FcRI signaling, we tested cells after exposure to several murine and rat mAbs of various Ig classes and subclasses as well as rabbit and goat polyclonal IgGs. As shown in Fig. 2A, rabbit IgG, which binds with high affinity to FcRI, supported signaling to the same extent as did hIgG1, whereas goat IgG neither bound nor affected response above the control. As shown in Fig. 2B, murine IgG2a and rat IgG2b mAbs are the subclasses that bind to FcRI, whereas murine mAbs of the IgA, IgE, and IgM classes, murine IgG1 and IgG2b mAbs, and rat IgG2a mAbs bind poorly or not at all. As shown, only the mAbs that bound enhanced O(2) production through FcRI. Some mAbs had antigen specificities for non-FcRI monocyte epitopes, but these had no apparent affect on O(2) production attributable to antigen (Fig. 2A, asterisks). Some exceptions were mIgG1 mAbs that recognize leukocyte function-associated antigens and an IgE mAb. These mAbs reduced FcRI-binding sites without affecting O(2) production. Overall, the results suggest that ligand occupancy by high affinity IgG enhanced FcRI-signaling for the oxidative burst.

Ligand-bound Structure

Hybrid IgGs contain only one IgG heavy chain capable of ligating FcRI(26) . To determine whether enhanced O(2) production requires both of the heavy chains in normal high affinity IgGs, hybrids with one or two IgG2a heavy chains were tested. Binding by the 2a/2a hybrid resulted in 100% of the enhancement of the hIgG1 control (Fig. 3). The 2a/1 and 2a/2b hybrids were less effective, perhaps due to an insufficient time for saturation binding, but were nevertheless capable of enhancing FcRI signaling. As the hybrid low affinity chain can interact with FcRII(29) , very short preincubation times were used, and duplicate assays were conducted in the presence of IV.3 Fab. As shown, IV.3 blocking of FcRII did not interfer with hybrid-enhanced FcRI signaling (Fig. 3), indicating that FcRII had not been involved.


Figure 3: Hybrid Abs enhance FcRI-mediated signaling. Cells were preincubated for 10 min with hIgG1 or hybrid murine mAbs comprising two IgG2a chains (2a/2a), one IgG2a and one IgG2b chain (2a/2b), or one IgG2a and one IgG1 chain (2a/1) (7 µg/ml each). Anti-FcRI F(ab`)(2) were added as in Fig. 2, and FcRI-mediated O(2) production was measured. The preincubations and assay were repeated in the presence of 25 µg/ml IV.3Fab. Data are expressed as a percent of the hIgG1 control.



The effect of hybrids suggested a ligated structure involving one receptor/IgG as opposed to a trimer composed of two receptors each binding to one heavy chain on IgG. For a more direct approach in establishing this ratio, we measured cell-associated 32, 32Fab, and 32F(ab`)(2), to establish the number of FcRI on cells; we measured cell-associated IgG2a, to establish the number of ligand binding sites/cell. The results of a FACS analysis (Table 1) show that IgG-binding sites equal the number of FcRI, indicating that the ligated structure must have a ratio of one FcRI/bound IgG.



IgG-binding Does Not Prime Cells or Increase FcRI Expression

The results above suggested that high affinity IgG increased the transmission of an excitatory signal. A plausible alternative was that IgG binding induced cell priming, a phenomenon whereby exposure to one reagent increases the oxidase response to a subsequent unrelated stimulant(30, 31) . To test this, we triggered hIgG1-preincubated cells through the receptor for fMLP with the result that fMLP-signaling was found unchanged by FcRI ligation (Fig. 4).


Figure 4: Effect of IgG on the fMLP-triggered respiratory burst. Cells were preincubated with medium (med), hIgG1, 32F(ab`)(2), or hIgG1 plus 32F(ab`)(2). They received 22F(ab`)(2) to trigger FcRI or fMLP (5 times 10M) to stimulate chemotactic receptors. O(2) production is expressed in mV.



Measurements by FACS analysis of the number of surface FcRI following a typical preincubation of cells with IgG revealed no significant increase in FcRI numbers (Table 2). Thus, neither cell-priming nor increased numbers of surface FcRI account for the increase in signaling.



Enhanced FcRI Signaling Is Not Due to a Specific Mode of Anti-FcRI Binding

As signal-enhancement may have been due to a specific mode of anti-FcRI binding, we questioned whether these Abs engaged ligated differently than unligated receptors or could desensitize the receptors. First, we examined the possibility that anti-FcRI might be capable of a ligand-blockable receptor desensitization. For this, O(2) production was measured after binding one anti-FcRI followed by ligand, or the reverse order, and cross-linking by the other anti-FcRI. As shown in Table 3, either order of binding elicited similar oxidative responses. That anti-FcRI did not form more monovalent complexes with unligated FcRI is also implied by this result. Furthermore, we speculated that ligation might involve a rearrangement involving rebinding of the first anti-FcRI. However, the addition of 32Fab to block rebinding by 32F(ab`)(2) did not block ligand-increased signaling (Table 3), suggesting that ligand-induced productive rebinding did not occur.



Second, the possibility that ligation might increase signaling through an increased rate of anti-FcRI binding was tested. Rates of binding depend on concentrations of the anti-FcRI antibodies(8, 9) . Since there is a linear relationship between the rates of binding of cross-linking antibody and O(2) generation(8, 9) , times of peak rates of response correspond to peak rates of binding. Therefore, peak times could be used as an index for the rate of binding. As shown in Table 4, peak times were changed by antibody concentration but not by IgG-ligation.



Third, FcRI were aggregated through anti-FcRI F(ab`)(2) plus sheep or goat anti-murine antibodies, or through 22Fab covalently conjugated multimers that cross-link FcRI and would have a different gross physical structure compared with 22F(ab`)(2). In each case, ligand-increased signaling was found (Table 5). These data show that enhancement was not due to a unique mode of bridging by 22F(ab`)(2). Collectively, the results indicate that enhancement was not due to a specific mode of anti-FcRI binding.



Cross-linking of Unligated FcRI Induces Receptor Aggregation and Internalization

The possibility that suboptimal signaling occurred because unligated FcRI could not be aggregated was assessed by measuring aggregation through induced internalization. Cells were exposed to the usual reagents, but cross-linking was performed using FITC-22F(ab`)(2). FACS analysis for internalized fluorescence demonstrated induced uptake of unligated FcRI as well as of ligand-occupied receptors (Table 6). The difference in uptake (0.15-fold) was minor compared with the effect of IgG on FcRI signaling (usually 3-5-fold), suggesting an indirect influence on uptake, possibly through signal-dependent changes in cytoplasmic milieu.



IgG Predisposes FcRI for Aggregation-dependent Signaling

FcRI signaling for O(2) production is transient (9) . Following cross-linking, receptors enter a surface accessible postsignaling stage(8) . Unligated FcRI, being aggregable, are also likely to enter a postsignaling stage following cross-linking. If IgG-binding predisposes FcRI to signal, it should have no effect on postcross-linked unligated receptors. Results consistant with this are shown in Fig. 5. In the control assay (Fig. 5A), cross-linking antibody was added at various times to discrete samples of cells to demonstrate their capacity to respond. In a second assay (Fig. 5B), the cells were exposed to IgG before or after the zero time addition of cross-linking anti-FcRI. As shown in Fig. 5B, IgG given to cells 3 min after FcRI were cross-linked produced a small reaction, but IgG given at later times gave responses identical those receiving no IgG. This implies that aggregated unligated receptors reached a postsignaling stage beyond that affected by IgG-binding. It confirms that aggregation-dependent signaling requires preoccupancy of the receptors.


Figure 5: IgG-binding prior to FcR-cross-linking produces the critical change in transductional activity. A, cells were preincubated with hIgG1 and 22F(ab`)(2), and individual samples of these cells were given 32F(ab`)(2) at 0, 3, 6, 9, or 12 min to stimulate O(2) production. One sample received no 32F(ab`)(2) (none). B, cells were preincubated in 22F(ab`)(2) for 10 min, and stimulated by the addition of 32F(ab`)(2). HIgG1 was added to individual samples of these cells at 0, 3, 6, 9, or 12 min, or no hIgG1 was added (none). Results are presented as rate of O(2) production, measured in mV/s and expressed as a percent of the optimum.



Dose Response of FcRI/IgG in Aggregates

We examined the question of whether each (FcRI/IgG) is capable of delivering a unitary excitation. Cells were incubated with saturating and subsaturating concentrations of IgG. The amount of ligand-bound to cells and their capacity to trigger respiratory bursts through mixtures of unligated and ligated-FcRI were measured. Data in Fig. 6on the dose dependence indicated that 2 µg of hIgG1/ml was both saturating for FcRI (Fig. 6B) and optimal for the FcRI-mediated respiratory burst (Fig. 6A). At the lower concentrations where ligated FcRI were diluted in aggregates by unligated receptors, O(2) production was not proportional to ligand-occupied receptors (Fig. 6C). Rather, production decreased exponentially with increasing numbers of unligated receptors. This indicated that the presence of unligated FcRI depressed responses to aggregated ligated receptors, and suggested that there was no trans-activation of unligated FcRI by ligated receptors, at least for O(2) signaling.


Figure 6: Effect of IgG concentration on aggregation-dependent FcRI signaling. In sequential experiments, cells were preincubated in O(2) assay medium containing 0, 0.06, 0.125, 0.25, 0.5, 1, 2, and 4 µg/ml hIgG1 (opencircles) or 0.7, 7, 23, 70, 230, and 7000 µg/ml hIgG1 (filledcircles). Samples in each were assayed for FcR-mediated respiratory bursts (A) and hIgG1-binding (B). B was measured by FACS analysis following an incubation with FITC-conjugated anti-hIgG antibody; data is expressed as the mean fluorescence intensity of cells. C, data were calculated from results in A and B to determine O(2) production as a function of hIgG1-binding. On a log plot, data falls on a straight line indicating that O(2) production/ligated receptor decreases by a simple exponential function (not shown). The saturation (SAT'N) point for IgG1-binding is indicated by an arrow. Data represent the mean ± the half-range of seven assays.



Phosphorylation of FcRI -Chains and Association of Syk

Since aggregation controls transmembrane signaling by clustering FcRI -chains inducing their tyrosine phosphorylation (15, 18) , we examined the effect of IgG on this activity. Following cross-linking, the receptors were precipitated, and their co-precipitating -chains were analyzed by anti-phosphotyrosine and anti--chain immunoblot. As shown in Fig. 7, increased numbers of phosphorylated -chains were identified in aggregates of FcRI/IgG compared with the unligated clusters. Receptor/subunit recovery was assessed through anti- immunoblot, which demonstrated that ligated receptors, aggregated or not, precipitated less efficiently. Experiments with similar recoveries revealed even larger differences in phospho- (not shown). As shown, phospho- was not detected in ligated, unaggregated FcRI. Interestingly, a 72-kDa tyrosine phosphorylated protein (Fig. 7A) also associating more extensively with FcRI/IgG aggregates was found through immunoblotting (Fig. 7B) to contain pp72Syk. (^2)These results show that IgG directly affected the receptor by increasing phosphorylation of -chains and the association of tyrosine phosphorylated pp72Syk.


Figure 7: Effect of IgG on -chain phosphorylation and the co-precipitation of pp72Syk. A, cells preincubated with or without hIgG1 were incubated in O(2) assay medium containing 22F(ab`)(2) or 22F(ab`)(2) and 32F(ab`)(2). After 5 min at 37 °C, the cells were centrifuged through cold PBS/100 uM Na(3)VO(4) (1 ml) and solubilized in digitonin lysis buffer. The receptors were precipitated by GAMk-AB1 beads, separated by nonreducing SDS-polyacrylamide gel electrophoresis, transferred, and analyzed by immunoblot using anti-phosphotyrosine (anti-PY). The membrane was stripped and reblotted with anti--chain (anti-) antibody. Blots were developed using horseradish peroxidase-conjugated second antibody and ECL. Phospho-2 is indicated by brackets. The anti--reactive 18-22-kDa proteins beneath phospho- are unphosphorylated -chain dimers. Arrow indicates pp72. B, anti-PY and anti-Syk immunoblots of 72-kDa proteins that co-precipitated with cross-linked FcRI.




DISCUSSION

In this report, we demonstrate that signaling resulting in activation of the NADPH oxidase pathway and involving the tyrosine phosphorylation of -chains on FcRI is markedly increased by IgG-Fc interaction with the receptor ligand binding site. To show the effect on FcRI-mediated NADPH oxidase activity, we triggered through FcRI on monocytic cells exposed to a variety of IgGs and found that all IgGs that bound to IFN--treated U937-10.6 monocytic cells, including rabbit, human subclasses 1 and 3, murine subclass 2a, and rat subclass 2b, increased the FcRI-mediated production of O(2). Low affinity IgGs and IgA, IgM, and IgE had no effect. Binding to some leukocyte integrins caused slight FcRI blocking but did not increase FcRI signaling.

Significantly, binding by IgG had a direct effect on the receptor in that it increased the aggregation-dependent tyrosine phosphorylation of FcRI -chains(15, 18) . This was accompanied by the binding to activated FcRI of a 72-kDa tyrosine phosphoprotein, identified as pp72Syk. This result is consistent with recent studies showing that Syk binds to tyrosine phosphorylated -chains in cells stimulated through FcRI(18) , to -chains in activated FcRI complexes(21, 32) , and to homologous tyrosine-phosphorylated motifs in activated B-cell receptor complexes(33) . We propose that IgG-binding creates a change in availability of FcRI -chains that permits aggregation-dependent tyrosine phosphorylation of -chain motifs and recruitment of pp72Syk. Another possibility is that ligand binding influences the affinity of the -subunits for associated Src kinases (17) , perhaps modifying the kinetics of reactions in aggregates.

This study also shows a correlation between -phosphorylation, pp72Syk association, and O(2) production, suggesting a link between FcRI-associated tyrosine kinases and the activation of the NADPH oxidase pathway.

Experiments to elucide possible effects of IgG other than on signaling through FcRI demonstrated that enhancement was not due to global cell-priming, because triggering through fMLP was unaffected. It was not due to an increase in the numbers of surface FcRI nor to specific modes of cross-linking by anti-FcRI antibodies. It was also not due to a lack of aggregability of unligated FcRI. These negative results help to support the proposed hypothesis that aggregation without ligand is transductionally insufficient.

The IFN--induced receptors on U937-10.6 cells appeared somewhat less IgG-Fc-dependent than those on monocytes. However, induced receptors on premonocytic U937 cells were not tested after a comparable expression time because several days of additional culture are required to develop an oxidative capacity in the tumor cells(23) . Furthermore, greater numbers of FcRI are expressed through induction on the U937-10.6 subclone. Thus, age or number (or some other factor) could have converted a minority of receptors to what may be an easily achieved signal-efficient configuration. Regardless of the reason, characterizing newly expressed normal monocyte FcRI as IgG-Fc-dependent strengthens our conclusions on the importance of Fc to FcRI signaling.

The nature of the improved signaling is not yet understood. However, several observations were made that partially characterize the ligand/receptor relationship. (i) IgG that was bound to cells was not itself cross-linked, making it unlikely that a mechanical strain altered the receptor and more likely that the interaction with IgG-Fc perse brought about relevant changes. (ii) Concentrations of IgG optimizing transductional activity were identical to those saturating FcRI, supporting the biochemical linkage and the specificity of the reaction. (iii) This interaction in unaggregated, signal-quiescent FcRI did not increase phosphorylation of . Therefore, potentiation was not accompanied by an obvious increase in activity of protein-tyrosine kinases associating with -chains(17) . However, binding could have increased protein-tyrosine kinase affinity, a point presently under investigation. (iv) When ligand was bound after receptor cross-linking, there was no return to oxidase activation. This outcome is consistant with the view that, with or without ligand, aggregation provides only a transient opportunity (interval/receptor) for transductional activity (8, 9) and that the ligand is effecting a change within this window. (v) At various subsaturating IgG concentrations, anti-FcRI cross-linking must have produced mixed aggregates of ligated and unligated FcRI, and in these cases, O(2) production decreased exponentially with the decrease in ligand-occupied receptors (Fig. 6C). Evidently, the unligated receptors were not only refractory as triggers but seemed obstructive to efficient signaling. The surprising implication is that the alteration in ligated FcRI seems not to have been transmitted to proximate unligated FcRI. If the alteration were a matter of affinity or availability of Src kinases on -chains, it is possible that the kinase concentration was diluted below a critical level for a trans-activation of the unligated FcRI. In any case, this evidence indicates that ligated and unligated FcRI are markedly different in ways that are critical and specific to transductional activity.

A further characterization using hybrid antibodies indicated that signal-enhancement was achieved through the binding of IgG containing only one high affinity heavy chain capable of binding FcRI. This evidence alone would not have excluded the possibility of hybrid low affinity chain engagement of a second FcRI or an FcRII. However, we made direct measurements that show that the potentiated FcRI structure has a ratio of one receptor/hIgG1 ligand. This indicated that the other heavy chain on hIgG1 does not engage with high affinity the ligand-binding site on a second FcRI. Additionally, measured with IV.3Fab as a block, the second heavy chain does not occupy the ligand binding site on FcRII. Therefore, as predicted by the hybrids, only one of the two heavy chains of IgG binds in a manner sufficient for potentiation. Unlike the ligand-induced dimerization characteristic of several cytokine receptor family members(34) , monomer ligand does not dimerize FcRI.

The results of this study do not alter the general view that aggregation, not a ligand-induced physical change in FcR, is the key event in transmembrane signaling(35) . But in addition to that, IgG-Fc binding must alter FcRI such that those events normally involved in aggregation-dependent signal transduction can occur. It is our speculation on this point that Fc binding stabilizes an activatable configuration of the receptor, one that may be present to varying extents in the nonphysiologic conditions of ligand-free cell culture. Fc region involvement other than in passive mediation of myelocytic antigen recognition has been suggested by Brown and Koshland(36, 37) , and some distinctions in FcR-mediated cytotoxicity due to hIgG-Fc in subclasses 1 and 3 have also been described(38, 39) . Physicochemical studies have shown that coupling of IgG to FcRI and IgE to FcRI induces conformational alterations in the ligands (40) and possibly in FcRI during an affinity shift(41) . Physical distances between FcR in aggregates (42) and interactions with cytoskeletal proteins may also be important(43) . Whether ligation changes the configuration of FcRI and how binding is communicated to cytoplasmic components will require further study.

In summary, the results in this report provide evidence that FcRI is altered by IgG-Fc interaction with the FcRI ligand-binding site and that this alteration permits efficient signaling of the oxidative inflammatory response. Phosphorylation of associated -chains and association by pp72Syk kinase were also increased by ligation of the receptors.


FOOTNOTES

*
This work was supported by Grant AI29455 from the National Institutes of Health (to L. C. P.) and by a grant from the Hitchcock Foundation, Hanover, NH. Flow cytometry was supported in part by the Core Grant of the Norris Cotton Cancer Center (CA23108). 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.

(^1)
The abbreviations used are: FcR, Fc receptors; FcRI, high affinity Type I Fc receptor; FcRI, high affinity Fc receptor; IgG, immunoglobulin G; O(2), superoxide; IFN-, interferon ; FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate.

(^2)
L. C. Pfefferkorn and S. L. Swink, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Clark L. Anderson, Ohio State University College of Medicine for many stimulating discussions.


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