Cross-linking of the high affinity Fc receptor for human immunoglobulin G1 triggers transient activation of NADPH oxidase activity. Continuous oxidase activation requires continuous de novo receptor cross-linking.

Cross-linking of the high affinity Fc receptor for human immunoglobulin G1 (Fc gamma RI) on U937 cells triggered superoxide anion (O-2) release. This was accomplished by the binding of an Fc gamma RI-specific monoclonal antibody, mAb 32, followed by cross-linking of the mAb on the cell with anti-mouse IgG F(ab')2 by Fc gamma RI-specific mAbs 32 and 22 used as an equimolar mixture or by Fc gamma RI-specific mAb 197 (a murine IgG2a and thus a multivalent ligand for Fc gamma RI) alone. At subsaturating concentrations of the Fc gamma RI-cross-linking ligands, O2- generation was continuous over relatively long intervals. However, saturating concentrations triggered an often substantial but always transient O2- burst. This transient burst of oxidase activity ceased with maximal ligand accumulation on the cell. Cells in which oxidase activity had ceased could be restimulated using phorbol 12-myristate 13-acetate or aggregated human IgG1, indicating that cessation of O2- generation was not due to a generalized exhaustion or inhibition of the NADPH oxidase pathway. Cells incubated in subsaturating concentrations of cross-linking antibodies continued to release O2- until binding of the ligand ceased. In addition, the rates of O2- production and ligand accumulation were the same. Thus, continuous O2- production appeared to be dependent upon continuous de novo formation of cross-linked and activated Fc gamma RI. Furthermore, the mol of O2- released in response to Fc gamma RI cross-linking by the multivalent ligand mAb 197 were directly proportional to the mol of mAb bound over a range of saturating and subsaturating concentrations. This evidence suggests a quantal relationship between each Fc gamma RI activated (cross-linked) and the resultant oxidase activity and supports a "rate" model for the activation of this response. Thus, each Fc gamma RI entering the pool of activated receptors probably makes a unitary contribution to the signal. An additional finding showed that cross-linked Fc gamma RI became associated with the cell cytoskeleton and that this association was also transient. Dissociation of Fc gamma RI from its cytoskeletal attachment occurred well after cessation of O2- production.


Cross-linking of the High Affinity Fc Receptor for Human Immunoglobulin G1 Triggers Transient Activation of NADPH Oxidase Activity
Cross-linking of the high affinity Fc receptor for human immunoglobulin G1 (FcyRI) on U937 cells triggered superoxide anion (0;) release. This was accomplished by the binding of an FcyRI-specific monoclonal antibody, mAb 32, followed by cross-linking of the mAb on the cell with anti-mouse IgG F(ab')z by Fc-yRIspecific mAbs 32 and 22 used as an equimolar mixture or by FcyRI-specific mAb 197 (a murine IgG2a and thus a multivalent ligand for FcyRI) alone. At subsaturating concentrations of the FcyRI-cross-linking ligands, 0; generation was continuous over relatively long intervals. However, saturating concentrations triggered an often substantial but always transient 0; burst. This transient burst of oxidase activity ceased with maximal ligand accumulation on the cell. Cells in which oxidase activity had ceased could be restimulated using phorbol 12-myristate 13-acetate or aggregated human IgG1, indicating that cessation of 0; generation was not due to a generalized exhaustion or inhibition of the NADPH oxidase pathway. Cells incubated in subsaturating concentrations of cross-linking antibodies continued to release 0; until binding of the ligand ceased. In addition, the rates of 0; production and ligand accumulation were the same. Thus, continuous 0; production appeared to be dependent upon continuous de novo formation of cross-linked and activated FcyRI. Furthermore, the mol of 0; released in response to FcyRI cross-linking by the multivalent ligand mAb 197 were directly proportional to the mol of mAb bound over a range of saturating and subsaturating concentrations. This evidence suggests a quantal relationship between each FcyRI activated (crosslinked) and the resultant oxidase activity and supports a "rate" model for the activation of this response. Thus, each FcyRI entering the pool of activated receptors probably makes a unitary contribution to the signal. An additional finding showed that cross-linked FcyRI became associated with the cell cytoskeleton and that this association was also transient. Dissociation of FcyRI from its cytoskeletnl attachment occurred well after cessation of 0; production. * This work was supported in part by National Institutes of Health Grants A119053 and CA44794 (to M. W. F.) and by a grant from the Hitchcock Foundation (to L. C. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Supported by a postdoctoral fellowship of the Arthritis Foundation during the course of these studies. To whom correspondence should be addressed.
The high affinity Fc receptor for human IgGl (FcyRI)' is found on monocytes, macrophages, and to a lesser extent, on interferon y-treated neutrophils (1,2). This receptor has been shown to mediate some of the responses characteristic of FcyR-stimulated phagocytes, including superoxide anion (0;) production by monocytes and by differentiated parent (3) and mutant cells' of the myelomonocytic cell line, U937. However, because the receptors have to be aggregated by multivalent ligands in order to trigger any of the FcyRlmediated responses, it has been difficult to assess the role of individual receptors in FcyRI activation of the NADPH oxidase. In addition, it has been difficult to determine whether the rates of oxidase activity triggered through FcyRI were a result of continuous receptor signaling by ligand-occupied receptors or whether the rates were due to a transient activation of the oxidase which continued by virtue of continued de nouo formation of receptor clusters. As part of our investigation into the relationship between the FcyRI activation and the activation of NADPH oxidase activity, we have used high affinity monoclonal antibodies (mAbs) specific for FcyRI, sometimes coupled with the use of second, anti-mouse IgG F(ab')', to cross-link the pool of FcyRI and stimulate 0; generation. Three FcyRI-specific mAbs were used. mAbs 32 (3) and 223 are of the mouse subclass IgGl and react with FcyRI at different epitopes and at epitopes distinct from the ligand-binding site of this receptor. mAb 197 is a mouse lgG2a antibody that binds with high affinity through its Fc region to the Fc-binding site of FcyRl (4) and which binds through its Fab region to an epitope outside of the Fc-binding site on FcyRI.3 Based on the binding characteristics of these antibodies, it seemed likely that a mixture of mAbs 32 and 22, or mAb 197 alone, would cross-link FcyRI into lattices and induce receptor-mediated functions. We have measured the kinetics of the binding of fluorescein-conjugated anti-FcyRlspecific monoclonal antibodies and the kinetics of the resultant oxidase response. Here, we report on the transience of FcyRI-activated oxidase activity and on the requirement for continuous receptor cross-linking to sustain oxidase activity.

Cell Preparation
A12.13 cells, high expression mutants of U937 cells for FcyRI and 0;: were incubated with 100 units/ml recombinant human interferon y (a gift from Genentech, San Francisco) in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (GIBCO) for 4-5 days before use.

mAb Preparation
All mAbs were purified from ascites by high pressure liquid chromatography separation on a DEAE-5PW column and by chromatographic separation on a protein A-Sepharose column.

Methods for Cross-linking FcyRI by the Use of
Cross-linking Antibodies Indirect Cross-linking-Cells were incubated with 10 pg/ml mAb 32 in RPMI/fetal calf serum containing 20 mM Hepes at 4 "C for 60 min. mAb 32-coated cells were washed three times. FcyRI'mAb 32 complexes on the coated cells were cross-linked with various concentrations of goat anti-mouse IgG F(ab')z (gam F(ab')A (Cappel, West Chester, PA), as indicated in the text.
Direct Cross-linking, Method I-Cells were incubated with various concentrations of an equimolar mixture of FcyRI-specific mAbs 32 and 22. mAbs 32 and 22, both mouse antibodies of the IgGl isotype, bind two different epitopes on FcyRI through the mAb Fab region^.^ Direct Cross-linking, Method 2-Cells were incubated with various concentrations of FcyRI-specific mAb 197, a murine IgG2a, which binds an epitope on FcyRI through its Fab regions and, with high affinity, the Fc-binding site of FcyRI through its Fc-binding domain? Microscopic observation indicated that incubation of cells with mAbs 32 and 22 or mAb 197 did not cause cell aggregation.

Superoxide Anion Assay
The method that was used to measure 0; is a modification of the method of Babior et al. (5). Cells were washed and preincubated in 0; assay medium (20 mM Hepes buffered with 10 mM KOH, 137 mM NaC1, 1 mM MgC12, 1 mM CaC12, pH 7.45) (HBS), containing 5.5 mM glucose and 200 mM KCN (6). Cell suspensions were mixed with ferricytochrome c (cytochrome c; type 111, Sigma) and stimulant (FcyRI-cross-linking antibodies or other stimulatory agents) or medium to final concentrations: cytochrome c, 0.5 mg/ml; cells, 106/ml; and stimulant, as indicated in the text, all in a volume of 1-1.5 ml. Samples of 150-200 p1 were taken at various intervals into a 96-well plate. Reduced cytochrome e in the samples was measured by absorption difference at 550 nm using a Dynatech MR 700 well-plate reader set in a dual wavelength mode. Values obtained at the reference wavelength (630 nm) and from control incubations lacking stimulant were subtracted. An extinction coefficient of AbS0 = 1.46 X lo4 M" cm", established previously for the 10-nm bandwidth of the Dynatech 550-nm filter (Footnote 2; method in Ref. 7), was used to quantitate reduced cytochrome c. In an alternative procedure, assay volumes of 6 ml were used, and 1-ml samples were centrifuged at various times. Reduced cytochrome c in 300-p1 volumes of cell-free supernatant was measured with a well-plate reader, as above. In some experiments, 20 mM NHdC1 was substituted for 20 mM NaCl in the 0; assay medium, as indicated in this section in order to inhibit lysosomal degradation of internalized cross-linking antibody. Evidence for the inhibition of reoxidation of reduced cytochrome c by KCN, an inhibitor of mitochondrial cytochrome c oxidase, and the quantitation of reduction is described under "Results."

Measurement of Cell-associated FcyRI-cross-linking Antibody
Cells were stimulated to generate 0; by directly or indirectly crosslinking FcrRI with cross-linking antibody. To measure the amount of cross-linking antibody associated with the cells, FITC-conjugated antibody was used in place of the unconjugated stimulants. Samples containing 1 0 ' cells obtained from the 0; production assay were washed three times with HBS, then fixed with HBS, 1% paraformaldehyde. Cells (5000 per assay point) were analyzed by flow cytometry with a cytofluorograph system 50H and system 2150 computer (Orthodiagnostic Systems, Westwood, MA). Excitation was with 300 milliwatts of 488-nm light from an argon ion laser. Green fluorescence was detected through a 530-nm long pass filter, and the mean fluorescence intensity (MFI) was computed from data expressed on a linear fluorescence scale of 0-1000. Autofluorescence was subtracted.
The following cross-linking antibodies were labeled with FITC: mAb 32, mAb F-32; mAb 197, mAb F-197; and F(ab'), of goat (Fgam) or rabbit anti-mouse Ab. FITC-labeled mAb 32 was mixed with unconjugated mAb 22 for all binding studies using F-32/22 mixtures or was used as the precoating antibody in some experiments in which crosslinked F-32 .FcyRI complexes were cross-linked using unlabeled gam F(ab'),.

Measurement of Cytoskebton-associated FcyRI
FcyRI were saturated with mAb F-32 at 4 "C. Cells were washed then incubated with gam F(ab')z in 0; assay medium. To determine the extent of cytoskeletal attachment, samples of cells were washed with phosphate-buffered saline containing 1 mg/ml bovine serum albumin, then with phosphate-buffered saline. Volumes of 50 pl containing 2 X 10' cells were added to 1 ml of extraction buffer: 10 mM Tris, pH 7.4, 150 mM NaCI, 0.5% Nonidet P-40, 0.1% purified bovine serum albumin for 5 min at 4 "C (8). Detergent-insoluble material (cytoskeletons) was sedimented at 150 X g for 5 min, washed twice with extraction buffer, and fixed with 1% paraformaldehyde in a volume of 0.2 ml. Cytoskeletons were analyzed by flow cytometry as described above. Cytoskeleton-associated FcyRI is expressed as the mean fluorescence intensity of 2000 cytoskeletons per data point after subtraction of autofluorescence.

Kinetics of 0: Generation Stimulated by Cross-linking 32.
FcyRZ Complexes with Various Concentrations of Anti-mouse ZgG Antibody-Since a functional consequence of receptor cross-linking is the activation of the NADPH oxidase, 0; production could be used as a functional measurement of receptor cross-linking. To establish the optimal effective concentration of cross-linking antibody with respect to the activation of the NADPH oxidase, FcyRI on differentiated A12.13 cells were exposed to a saturating concentration of an FcyRI-specific monoclonal antibody, mAb 32. FcyRI-bound mAb 32 were cross-linked by various concentrations of gam F(ab')z. Fig. 1A shows the time course of 0; generation that was triggered by different amounts of second antibody. 0; production at 3, 9, and 40 min is shown in Fig. 1B as a function of the concentration of gam F(ab')z. mAb 32 alone did not trigger 0; release. These results indicate that there were two second antibody concentration-dependent phenomena: (a) a linear and concentration-dependent release of 0; over 90 min triggered by concentrations of second antibody in the range of 0.1-1 pg/ml with an "optimal" effective concentration at approximately 0.2 rg/ml; and (b) a burst of 0; released within the first 3 min of stimulation by concentrations higher than 2 pg/ml at an "optimal" effective concentration of approximately 20 pg/ml. Furthermore, 0; production ( Fig. 1B) was dependent on the concentration of crosslinking antibody prior to cessation of 0; production. High levels of cytochrome c reduction at the later time points indicate that cessation of 0; generation was not due to a depletion of oxidized cytochrome c.
To determine whether the second antibody used to crosslink mAb 32 on cells saturated all of the binding sites on mAb 32 during the brief burst of oxidase activity, mAb 32. FcyRI complexes were cross-linked with a high concentration of gam F(ab')*. This cross-linking produced an oxidative response ( Fig. 2A) and at the same time rapidly saturated epitopes on the receptor-bound mAb 32 (Fig. 2B). Therefore, the oxidative burst triggered by the high concentration of second antibody coincided with saturation of FcyRI-bound mAb 32.
Kinetics of 0; Generation Stimulated by Various Concentrations of FcyRZ-specific mAbs-To determine whether the bimodal response to second antibody cross-linking ( Fig. 1B) was characteristic of the normal cell response to cross-linking of FcyRI, the time course of 0; generation triggered by alternate FcyRI-cross-linking antibodies was measured. Ear- NADPH Oxidase Activity lier studies have shown that Fab fragments of FcyRI-specific mAbs 32 and 22 bind different epitopes on F c~R I .~ Although neither mAb alone stimulated 0; production in A12.13 cells, 0; release was triggered by an equimolar mixture of mAbs 32 and 22 (Fig. 3, A and B). Concentrations of 0.1-0.5 pg/ml mAbs 32/22 stimulated continuous 0; release over an 80-min incubation. Concentrations of 2-20 pg/ml mAbs triggered transient 0; release with little or no further activity after 6 min. As shown in Fig. 3B, the relationship of concentration to accumulated reduced cytochrome c was similar to that obtained by gam F(ab')2 cross-linking of mAb 32. FcRI complexes (Fig. 1B). Similar results were obtained when mAb 22 was added after binding mAb 32 to FcyRI on the cells ( Fig. 1. Control and mAb 32precoated cells were incubated in an 0; production assay f 1 0 pg/ml gam F(ab'),. The cells were centrifuged, and the reduced cytochrome (Cyt) c in the supernatant was measured ( A ) . The cell pellets were exposed at 4 "C for 60 min to F-rabbit am (FZTC-am) in the presence of 1 mg/ml hIgGl ( B ) to detect bound mAb 32 unblocked by the F(ab'), gam. Additional control and mAb32-precoated cells were incubated in 0; generation assay medium with 10 pg/ml FITC-gam F(ab'), at 37 "C for 2 min (C, 1st and 3rd burs) or 37 "C for 15 min ( C , 2nd and 4th bars). These cells were washed immediately after the incubation, and the 0; generation assay medium was discarded. Fluorescence associated with unfixed cells was measured by cytofluorography, as described under "Experimental Procedures," and is expressed as the mean fluorescence intensity. Variation between duplicate samples did not exceed 5% in this assay. Following the cessation of 0; production, oxidase activity was restimulated with the addition of phorbol 12-myristate 13acetate or aggregated IgG1. Second reagent stimulation reactivated the oxidase system in the prestimulated cells to levels comparable to those of the control cells (Fig. 4). This shows that neither the phorbol 12-myristate 13-acetate nor surface receptor-mediated stimulation of the oxidase was blocked in cells that had been triggered by high concentrations of crosslinking antibody. Therefore, cessation of 0; does not involve a generalized inhibition of the oxidase.
Effect of KCN on Early Cessation of 0; Generation-Since 0; generation requires ATP, we measured the kinetics of 0; release from mAb 32-precoated cells that were stimulated with second antibody in the presence or absence of KCN. The results (Fig. 5) indicated that maximal levels of 0; release were not altered by the presence of KCN. More importantly, reoxidation of the cytochrome c that was reduced during the early burst of oxidase activity or during low but prolonged oxidase activity was effectively blocked in assays containing KCN. Thus, inclusion of KCN in the assay prevented reoxidation of reduced cytochrome without affecting its rate of reduction. The interval over which reduced cytochrome c was stabilized was sufficient to make cytochrome c reduction in the presence of KCN a quantitative measure of cumulative 0; levels.
Comparison   5. Quantitation of 0; generation: effect of KCN on the rate and amount of cytochrome (Cyt) c reduction. Cells were exposed to mAb 32, washed, and stimulated in 0; assay medium with 0.25 pg/ml (0) or 5 pg/ml (0) gam F(ab'), as in Fig. 1. Included in the 0; assay medium was 500 p~ KCN (-) or no inhibitor (---). Values obtained in the absence of second antibody were subtracted. Shown is a representative example of four such experiments.

197-We tested cells on which FcyRI had been cross-linked
for the kinetics of 0; generation and the kinetics of the binding by mAb 197. This was carried out at various concentrations of the cross-linking mAb. Fig. 6, A-C, shows the results of mAb F-197 binding to the cells during stimulation of the oxidase. The three concentrations selected to stimulate the cells are from the high, low, and intermediate concentra- tions and give representative rates of binding and 0; release for those concentration ranges. In most cases, there was an apparently linear relationship between the two activities. The exception was that at very low concentrations of cross-linking mAbs, there was a delay in 0; production following the binding of mAb to the cells. Fig. 6D shows cumulative data from the binding and stimulation experiments in which mAb F-197 or mAbs F-32/22 had been used as cross-linking antibody. We show by these comparisons that (a) 0; generation ceased when there was no further accumulation of crosslinking antibody on the cell; and ( b ) that 0; generation continued as long as mAb continued to accumulate on the cells.
In addition, we found that after cells had been incubated in cross-linking antibody for a period of time, the accumulation of the antibody on the cells stopped. This accumulation of antibody was measured for each concentration of cross-linking antibody in several experiments (Fig. 7). Our data indicated that the total amount of antibody that accumulated on the cells was dependent on the concentration of free antibody at the start of the incubation and presumably on the avidity of its binding to FcyRI. The data also show that concentrations of antibody in the high range saturated FcyRI-binding sites and that the tested low concentrations of mAbs were well below those required to saturate available receptors.
To establish whether there was any difference in the relative effectiveness of bound mAb 197 and mAbs 32/22 to stimulate the oxidase, we compared the total amount of accumulated cell-associated mAb with the total amount of accumulated 0;. We found that over the range of FcrRIsaturating and -subsaturating concentrations of mAb 197 (Fig.  7), stimulation by this antibody produced a set ratio of total 0; evolved per unit of mAb bound (Table I amount of mAb F-32 bound (Fig. 7) but decreased the amount of 0; produced per unit of mAb F-32 bound (Table I).
To measure the effect of hIgG at different times during stimulation by mAb 197, cells were preincubated in a 500-fold excess of hIgG, and the kinetics of 0, generation and mAb binding were determined. 0; generation was initially blocked (Fig. 8 A ) and the mAb 197-binding rate reduced (Fig. 8 B )   cated by only a 5% decrease in the numbers of surface FcyRI.
Cross-linking Antibody-dependent Association with Cell Cytoskeleton-Another phenomenon associated with receptormediated transient activation of the oxidase pathway is the association of receptors with the cell cytoskeleton (9). To determine whether FcyRI became associated with the cell cytoskeleton, we measured the amount of FcrRI in the detergent-insoluble fraction of lysed cells on which the receptors had been cross-linked. Fluorescein-labeled mAb 32 was bound to FcyRI and then cross-linked with second antibody. FcyRI that bound mAb F-32 but were not cross-linked by second antibody were not found in the detergent-insoluble fraction. As shown in Fig. 9A, 5 pg/ml second antibody triggered a transient burst of 0; and a transient association of detectable levels of bound mAb F-32 with cell cytoskeletons (Fig. 9C). Dissociation of mAb 32 from the detergent-insoluble material occurred when 72% of total bound mAb was still cell associated (Fig. 9B). The amount of FcyRI association with the cell cytoskeleton was considerably less when the concentration of second antibody was 0.25 pg/ml (Fig. 9C). Th' 1s contration was less stimulatory to the cells (Fig. 9A). The results show that the transient 0; burst preceded cytoskeletal dissociation and coincided with cytoskeletal association of FcyRI-bound mAb F-32.

DISCUSSION
FcyRI-specific mAbs Trigger Oxidase Actiuity-The ability of antibodies to cross-link FcyRI has been defined by the induction of functions known to be triggered by multivalent FcyR ligands. The manner in which they interact with FcyRI to initiate 0; production has been determined using three different anti-FcyRI mAbs alone and in combination and by examining cells incubated with mAbs lacking specific domains required for cross-linking or in the presence of blocks of the receptor-binding sites. Binding by the F(ab')* of mAb 197 is insufficient to trigger 0; generation. In addition, hIgG1, which binds through its Fc domain to the ligand-binding site with the same high affinity as the murine IgG2a Fc domain of mAb 197 (4), temporarily blocks mAb 197-triggered 0; production. Therefore, it is probable that mAb 197 triggers oxidase activation by cross-linking FcyRI through the binding of Fc and one or both Fab domains. We suggest that Fc and Fab binding probably spans at least two receptors thereby linking numbers of FcyRI into a stimulatory receptor cluster. HIgGl also delays mAb 197-dependent FcyRI internaliza- antibodies, 32 and 22, is probably capable of spanning two receptors, as suggested by their ability to stimulate 0; production and internalization4 when both are present. It has been assumed that stimulation of cell response through surface receptors involves a change in the conformation of the receptor which is transmitted to the cytoplasmic domain, initiating signaling events (6,13). FcyRI may undergo a conformational change but that appears to be independent of ligand occupation and is supplanted or initiated by receptor aggregation.
Cross-linking Antibodies Trigger an Activation/Deactiuation of the NADPH Oxidase-As expected from reports published previously on the 0; response triggered by multivalent ligands such as opsonized particles (6), immune complexes (3), or aggregated hIgG1; low concentrations of cross-linking antibodies stimulated slow but continuous 0; production for 30-80 min. However, when 0; generation was triggered by high concentrations of cross-linking antibodies, a substantial but brief (<3 min) 0; response was the rule. The rapid burst followed by an abrupt cessation suggested that a high rate of oxidase activity was terminated by an oxidase deactivation. If receptor cross-linking activates FcyRI to signal the oxidative response for the interval that the receptors are "OCCUpied," the apparent deactivation could have been due to exhaustion of oxidase substrate, depletion of available oxidase, or a generalized inhibition of the oxidase pathway. The following evidence suggests that this is not the case.
(a) Regardless of the total output by the oxidase (i.e. -3 nmol of cytochrome c or 30 nmol/106 cells) cessation occurred during the burst. Therefore, cessation is probably not the result of oxidase substrate depletion. In addition, there was no evidence of any inhibitory effect of KCN on the kinetics of oxidase activation/deactivation. Thus, it was apparent that we had not altered cytoplasmic ATP replenishment or NADPH levels, which may have inadvertently produced an apparent deactivation.
( b ) Termination of oxidase activity took place in cells with demonstrably greater oxidative capacity. Therefore, cessation took place in the presence of additionally available but apparently unactivated oxidase molecules and was not due to oxidase exhaustion or desensitization.
(c) Additional stimulatory reagents reinitiated 0; production by cells that had undergone an FcyRI-mediated oxidative burst. These agents included phorbol12-myristate 13-acetate, which would be expected to activate protein kinase C (14)(15)(16), and aggregated IgG1, which would be expected to stimulate through the signal transduction pathways common to oxidasesignaling surface receptors. We have no direct evidence that the same molecules of oxidase deactivated by the first stimulus were reactivated by the second stimulatory reagents. However, resumption of oxidase activity in these cells indicated that cessation of 0; generation was not due to a generalized inhibition of the NADPH oxidase system. Transient Oxidase Activation during Cluster Formation-A comparison of the rates of binding of mAbs 197 and 32/22 to the rates of 0; generation revealed that at saturating concentrations, the time required for maximal accumulation of the mAbs on the cell coincided with the cessation of 0 2 production. At subsaturating concentrations of cross-linking mAbs, 0; production continued over the same prolonged interval as mAb binding. The initial lag in production seen only in the presence of the lowest concentrations of cross-linking antibodies may have been the result of a low rate of recruitment of receptor di-or trimers into larger stimulatory clusters. This evidence strongly suggests that continued 0; production is sustained by the continued binding of antibody which prob-ably causes continued de novo formation of stimulatory receptor clusters. Moreover, similarities in the rates of 0; production and of presumed cluster formation suggest a quantal relationship between activated receptors and the activation of the oxidase. Significantly, 0; production ceased when cross-linking mAb binding ceased, an indication that the activation event is probably transient.
Some published accounts of the deactivation of oxidase activity initiated through surface receptors (17)(18)(19)(20) show that deactivation is rapid but do not indicate the state of the receptors when oxidase activity terminated. In some reports, deactivation has been attributed to rapid loss of ligand from ligand-receptor complexes followed by a putative reversion of the receptors to the nonstimulatory configuration. However, our experiments with cross-linkers of FcyRI indicate that oxidase activation is terminated in spite of the continued cross-linked state of the receptor. Furthermore, termination occurs prior to cluster internali~ation.~ Therefore, continued occupancy of the FcyRI by cross-linking antibody does not dictate continued oxidase activation.
Novel Response to mAb 197-When binding equilibrium was reached, the high concentrations of cross-linking mAbs had saturated FcyRI, whereas the subsaturating concentrations had bound to an extent that reflects the concentration of available antibody and its avidity for the receptors. An unusual finding was that when one of the cross-linking antibodies, mAb 197, had accumulated to binding equilibrium, the cells had produced a constant ratio of O;/mol of bound antibody. The ratio was the same regardless of the final amount of cell-associated mAb. A constant stoichiometry at equilibrium was not the case for the mAb 32/22 mixture, which triggered the production of decreasing total amounts of O;/mol of bound mAb as saturating concentrations were approached. The difference may be due to the fact that mAbs 32/22 require twice the specific binding events of mAb 197 to produce a stimulatory combination, and the production of such a stimulatory combination may be less favored at high mAb 32/22 concentrations.
The reasons for bound mAb 197 triggering amounts of 0; production in proportion to the amount of mAb bound, without any apparent inhibitory effect by saturating concentrations of the antibody, are more subtle. We believe that the higher avidity of the mAb for FcyRI when it is bound through both its Fc and Fab domains may well prevent sustained monovalent Fc or Fab domain binding. If sustained monovalent binding is prevented, then excess mAb would be unable to block cross-linking and stimulation. In support of this explanation is the finding that hIgG1, which has the same affinity (21,22) for the receptor ligand-binding site as the Fc domain of murine IgG2a (4), only transiently blocks the ability of mAb 197 to stimulate cells. It is possible that Ab 197 is displacing the block. Thus, stimulation in the presence of the monovalent ligand for the Fc-binding site supports our conclusion that the multivalency of mAb 197 increases its apparent affinity for that site beyond that of monomeric Fc ligands and enables the antibody to cross-link most of the receptors. This phenomenon of rapid displacement from surface receptors of high affinity monomeric (more dissociable) ligands by multivalent (less dissociable) ligands has been described previously (23, 24).
Rate Versus Occupancy Theories of Receptor Activation-There are two theoretical models of receptor activation for signaling cell response. The occupancy theory holds that receptors are in an activated state for the duration of ligand occupancy and that this state promotes sustained activation of cell response. Therefore, at equilibrium binding, the rate of cell response should be highest. In the early 1960s, Paton proposed a rate model in which a rate of response would be determined by the rate of formation of ligand-receptor complexes (25,26). In this case, the ligand-binding event would trigger one quantum of excitation before inactivation of the ligand-receptor complex. Thus, the rate of response would be dependent on the rate of de novo formation of complexes. In a strict interpretation of a quantum of excitation, each activated receptor would elicit a set unit, or quantum, of response regardless of the number of receptors involved or when they became activated.
That such a quantal relationship between cross-linked, and therefore activated, FcyRI and oxidase activity may exist is suggested by two lines of evidence. The rapid reversibility of oxidase activation has been shown by numerous investigators (17-20, 27-29). Moreover, oxidase activation through the receptor for fMLP is transient (27,29-32) and possibly quantal (30). In the case of the fMLP receptor, where monovalent ligand interacts with monovalent receptor, we would expect constant ligand-related responses because each receptor would be expected to make a unitary contribution. It was not predicted that FcyRI aggregates of possibly varied size might also promote responses that are proportional to ligand binding and, in the case of mAb 197, stoichiometric. Collectively, their results and ours suggest common rules governing ligand-receptor performance and suggest that, clustered or not, each activated receptor may signal in a unitary manner.
Transient Association with the Cell Cytoskeleton-Our experiments revealed a transient association of cross-linked FcyRI with the cell cytoskeletons of A12.13 cells. At concentrations of cross-linking antibody saturating for the receptors, there was an apparently synchronous association and then dissociation of cross-linking antibody from the detergentinsoluble material. This dissociation appears to follow rather than coincide with cessation of oxidase activity. When FcyRI were cross-linked more slowly so that there would be a continuous de novo receptor activation (as indicated by continued 0; production), association was so brief that there was little cytoskeletal associated FcyRI at any one instant. This lack of accumulation of postactivated receptor on cytoskeletal actin gives some indication of the transience of this processing step. Actin association following signaling through FcyRI is consistent with the postsignaling processing events described for the fMLP receptors of neutrophils (9, 33-36).
Other Considerations Regarding Activation and Processing-Our observations of an apparent quantum of response argue that FcyRI is a rate receptor but do not reveal the molecular details about what terminates the activating event. The strict interpretation of the rate model presented by Van  Haastert (13) defines a transient activated state as inherent to the signaling step and independent of subsequent processing steps. If cross-linked FcyRI continued to signal until bound through an actin-binding protein to the cell cytoskeleton, this step could act to terminate signaling and to determine the duration of each quantum of signal. Jesaitis and colleagues (33, 34), describing the processing of the fMLP receptor, have shown some evidence favoring this possibility. The molecular events underlying the presumed signal termination of the fMLP receptor have yet to be elucidated, but, given the similarities with the FcrRI, the results of such studies may well have broad application.
Summary-Cross-linking of FcrRI by three different crosslinking strategies produced an activation then a deactivation of the NADPH oxidase pathway. Our results favor an explanation for deactivation which describes the relationship between activated FcyRI and activated oxidase as transient, dependent upon the continued formation of activated FcyRI for sustained oxidase activation, and one that produces a set quantum of 0; for each cross-linked FcyRI.