©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Heterotypic FcR Clusters Evoke a Synergistic Ca Response in Human Neutrophils (*)

Paula J. M. Vossebeld (1), Jan Kessler (1), Albert E. G. Kr. von dem Borne (1) (2), Dirk Roos (1), Arthur J. Verhoeven (1)(§)

From the (1) Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands and the (2) Department of Hematology, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Both Fc receptors on human neutrophils (FcRIIa and FcRIIIb) are capable of initiating signal transduction after multivalent cross-linking. However, immune complexes most likely activate neutrophils by a combined homotypic and heterotypic cross-linking of FcRs. We have investigated the effect of homotypic and heterotypic FcR cluster formation on changes in the intracellular free Ca concentration. Combined heterotypic and homotypic cluster formation resulted in a Ca response that was strongly enhanced as compared to the sum of both individual FcR responses. This synergistic response was caused by the formation of heterotypic clusters of FcRs and not by the simultaneous formation of homotypic clusters. This conclusion was supported by experiments with a bispecific antibody binding to both FcRIIa and FcRIIIb. The heterotypic FcR cross-linking results in efficient activation of Ca influx, probably caused by a more pronounced depletion of intracellular Ca stores. Stimulation with immune complexes also induced Ca influx in normal neutrophils, but not in FcRIIIb-deficient neutrophils. The synergism between both FcRs was also apparent in other responses of neutrophils, such as the activation of the respiratory burst. This study shows that the two different FcRs on neutrophils complement each other in mediating an important cellular response.


INTRODUCTION

The human polymorphonuclear neutrophil expresses two different types of receptors that can bind the Fc domain of IgG antibodies in immune complexes. These Fc receptors (FcRIIa and FcRIIIb)() play a key role in host defense mechanisms by linking the humoral immune response to the cell-mediated effector system. The FcIIa receptor is a 40-kDa transmembrane molecule with an expression of 10,000 to 20,000 molecules per neutrophil. The FcIIIb receptor is a heavily glycosylated protein with an apparent molecular mass of 50 to 80 kDa, linked via a glycosylphosphatidylinositol-anchor to the membrane; per neutrophil 100,000 to 200,000 molecules are expressed (1, 2) . The individual quantitative and qualitative role of each receptor in neutrophil activation has not yet been unraveled in detail. Multivalent cross-linking of FcRIIa clearly induces signal transduction in the neutrophil: a rise in [Ca], phagocytosis, degranulation, and the respiratory burst can be initiated via FcRIIa (3, 4, 5, 6, 7, 8, 9) .

The results of some studies suggest an inability of FcRIIIb to transduce signals independently of FcRIIa (3, 5, 8, 10, 11) . These results, together with the lack of transmembrane and cytosolic protein domains and the high expression level of this receptor on the cell surface, have led to the belief that FcRIIIb is principally a binding molecule that presents ligands to FcRIIa (3, 11) . However, several lines of evidence have emerged that point to a more extended role for FcRIIIb. Multivalent cross-linking of this receptor alone initiates, by still unknown mechanisms, signal transducing events such as membrane potential changes and an increase in [Ca](4, 12) , and can lead to actin filament assembly (13) . Moreover, several effector functions, such as killing of chicken erythrocytes coated with anti-FcRIII-Fab (14) , degranulation (15) , phagocytosis of ConA-opsonized erythrocytes (16) , and activation of the respiratory burst (4, 17, 18) , have been observed to be induced via FcRIIIb in neutrophils.

The ability of immune complexes to bind both to FcRIIa and FcRIIIb raises the possibility of interactions between the two receptors or the signal transduction elements connected to these receptors. Indirect evidence for such a cross-talk between FcRs on neutrophils has recently been obtained (3, 4, 19, 20, 21) . In the present study, we have investigated the effect of homotypic and heterotypic FcR cluster formation on changes in the intracellular free Ca concentration ([Ca]). To achieve controlled conditions of FcR cluster formation, monoclonal antibodies against FcRII and FcRIII were cross-linked under different conditions. We present evidence that heterotypic cross-linking of FcRIIa and FcRIIIb produces a synergistic Ca response in human neutrophils. Induction of Ca influx from the extracellular medium is important for this synergistic increase in [Ca]. Furthermore, we observed also synergism in other functional responses of neutrophils, such as the activation of the respiratory burst.


MATERIALS AND METHODS

Isolation of Neutrophils

Peripheral blood was obtained from healthy individuals and from a healthy FcRIIIb-negative donor, as described by Huizinga et al.(22) . Neutrophils were purified from buffy coats of 500 ml of blood anticoagulated in 0.4% (w/v) trisodium citrate and centrifuged through a Percoll layer with a specific gravity of 1.076 g/ml (1000 g, 18 min, 20 °C). Contaminating erythrocytes in the pellet fraction were removed by lysis in ice-cold buffer containing 155 mM NHCl, 10 mM KHCO, and 0.1 mM EDTA (pH 7.4). The neutrophils were washed twice in phosphate-buffered saline (PBS) and resuspended in incubation medium containing 132 mM NaCl, 6 mM KCl, 1 mM CaCl, 1 mM MgSO, 1.2 mM NaHPO, 20 mM Hepes, 5.5 mM glucose, and 0.5% (w/v) human serum albumin (pH 7.4). The purity of the neutrophils was more than 95%, the remaining cells were eosinophils.

Antibodies

The anti-human FcRIII mAb 3G8 (mIgG1) (23) and the anti-human FcRII mAb IV.3 (mIgG2b) (24) were purified from hybridoma culture supernatant by precipitation with 50% saturated ammonium sulfate and subsequent protein A affinity chromatography. The anti-major histocompatibility complex (MHC) class I mAb W6/32 (mIgG1) was purified from ascites fluid by protein A affinity chromatography.

F(ab`) fragments were prepared by digestion with 2% (w/w) pepsin at pH 3.7 for 3G8 and pH 4.0 for W6/32 for 16 h at 37 °C, followed by protein A affinity chromatography to remove free Fc fragments and intact antibodies.

Fab fragments were made by digestion with 4% (w/w) papain for 1.5 h at 37 °C in PBS containing 10 mM cysteine and 5 mM EDTA. The reaction was terminated by addition of 20 mM iodoacetamide. Protein A affinity chromatography was used to remove Fc fragments and intact antibodies. When F(ab`) and Fab fragments were checked on SDS-PAGE, intact antibodies or Fc fragments were not detectable.

Antibodies were biotinylated with biotin- N-hydroxysuccinimide ester (2 mg/mg IgG) for 4 h at room temperature. Free biotin was removed by dialysis against PBS. Cross-linking of antibodies was performed with polyclonal goat anti-mouse immunoglobulin (GAM) F(ab`) fragments against intact antibodies or against Fc domains of antibodies (Jackson Immunoresearch, West Grove, PA). Streptavidin (10 µg/ml) was used to cross-link biotinylated antibodies bound to neutrophils.

MAbs 3G8 and IV.3 were conjugated to fluorescein isothiocyanate (FITC) by incubation for 2 h at room temperature with FITC (4 times molar excess of FITC) at pH 9.5. Free FITC was removed by dialysis against PBS. All mAb were stored at 4 °C in PBS with 0.01% azide. Heat-aggregated IgG was freshly prepared for each experiment by incubating human IgG (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands) at 30 mg/ml for 30 min at 63 °C. Insoluble aggregates were removed by centrifugation at 10,000 g for 10 min.

Synthesis of Bispecific F(ab`) Antibody (BsAb) Recognizing FcRII and FcRIII

Fab fragments of IV.3 (3 mg/ml in PBS), prepared by digestion with papain as described above, were incubated for 5 h with regular shaking at room temperature with a 10-fold molar excess of the heterobifunctional cross-linker N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP) (Pierce) added from a stock solution in ethanol. To remove unbound SPDP, the reaction mixture was passed through a G-25 Sephadex column equilibrated with PBS. Fab`-SH fragments of 3G8 were prepared by reducing F(ab`) fragments (2.8 mg/ml in PBS), obtained by pepsin digestion as described above, with 15 mM 2-mercaptoethanol for 30 min at 30 °C. The reduced product was passed through a G-25 Sephadex column in PBS to remove the 2-mercaptoethanol and was immediately added to the IV.3 Fab-SPDP fragments. After the column step, a sample of 3G8 Fab`-SH was taken and incubated for 14 h to control for spontaneous reoxidation of the Fab`-SH fragments. Analysis on SDS-PAGE showed that this was not the case. The mixture of Fab`-SH and Fab-SPDP fragments was concentrated to one-third of the original volume in C30 Amicon microconcentrators (Amicon, Beverly, MA). After 14 h of incubation at room temperature, the mixture was passed through a fast protein liquid chromatograph Superose 12 column equilibrated with PBS. Appropriate fractions were pooled and analyzed on SDS-PAGE. The fraction containing dimers was taken for further characterization as bispecific antibody against FcRII and FcRIII (bsAb FcRIIxFcRIII).

Characterization of BsAb FcRIIxFcRIII

The fraction containing the bsAb FcRIIxFcRIII was first tested for its ability to inhibit the binding of the parent antibodies IV.3 and 3G8 to human neutrophils. For this purpose, purified neutrophils were fixed in PBS containing 1% (w/v) paraformaldehyde for 10 min at 4 °C. After washing in PBS containing 1% (w/v) bovine serum albumin, 2 10 cells were incubated with bsAb (3 µg/ml) or with a control mIgG1 antibody in the same concentration for 45 min at 4 °C. After washing, the cells were incubated with GAM-FITC (62.5 µg/ml) (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands), IV.3-FITC (2.5 µg/ml), or 3G8 F(ab`)-FITC (2.5 µg/ml) for 30 min at 4 °C. After another washing step, cell-associated fluorescence was measured in a flow cytometer (Becton Dickinson FACScan, Palo Alto, CA). Further characterization of the bsAb was performed with a Chinese hamster ovary (CHO) cell line transfected with human FcRIIIb cDNA (CHO cells) (25) . These cells did not express human FcRII. Wild type CHO cells (CHO cells) were used in these experiments as a negative control.

Measurements of the Cytosolic Free CaConcentration

Determination of [Ca]was performed as described before (26) . In short, neutrophils (2 10/ml in incubation medium) were loaded with indo-1 by incubation with 1 µM indo-1/AM (Molecular Probes, Eugene, OR) for 40 min at 37 °C. The neutrophils were washed and resuspended in incubation medium to 2 10/ml and kept in the dark at room temperature. Unless indicated otherwise, the cells loaded with indo-1 were diluted to 2 10/ml in incubation medium with 1 mM Ca and incubated with the appropriate antibodies for 5 min at 37 °C followed by washing and transfer to a cuvette. Fluorescence changes of the neutrophil suspension, magnetically stirred and kept at 37 °C, were monitored with a spectrofluorometer (model RF-540, Shimadzu Corporation, Kyoto, Japan), with 340 and 390 nm as excitation and emission wavelengths, respectively. To calibrate the indo-1 fluorescence (27) as a function of [Ca], all trapped indo-1 was saturated with Ca by addition of digitonin (10 µM), after which the indo-1 fluorescence was quenched with MnCl (0.5 mM). A dissociation constant of 250 nM for the indo-1Ca complex was used to calculate [Ca](28) .

For intracellular Ca measurements in the presence of EGTA, indo-1-loaded neutrophils were diluted to 2 10/ml just prior to stimulation in incubation medium (without CaCl) containing 1 mM EGTA. Before the calibration with digitonin, CaCl (2 mM) was added.

In some experiments, neutrophils were loaded with 10 µM BAPTA-AM (1,2-bis-( O-aminophenoxyl)ethane- N,N,N`,N`-tetraacetic acid) (Molecular Probes) as follows. Prewarmed neutrophils (2 10/ml in incubation medium) were first incubated with 1 µM indo-1/AM for 10 min at 37 °C, and then 10 µM BAPTA-AM was added. After 30 min, the cells were washed and resuspended in incubation medium without Ca (2 10/ml).

Assessment of Ca influx was carried out with the Mn quenching technique (29) . For this purpose, an emission wavelength of 446 nm instead of 390 nm was chosen, resulting in the complete absence of fluorescence changes upon addition of a Ca mobilizing stimulus (data not shown). For these experiments, the indo-1-loaded neutrophils were incubated in incubation medium with Ca (2 10/ml) for 5 min at 37 °C with the appropriate antibodies, washed, and resuspended (2 10/ml) in medium containing 0.2 mM CaCl (to favor Mn entry) (30) . Two min prior to addition of the stimulus, 0.5 mM MnCl was added to the indo-1-loaded neutrophils (2 10/ml) and fluorescence changes were recorded in time as described above. Except for the traces shown in Figs. 6 and 7, scanning of the fluorometer traces was performed followed by smoothing in the computer program Correldraw.

Measurement of the Respiratory Burst

Activation of the respiratory burst was measured with 1,2,3-dihydrorhodamine-loaded cells, as described (31, 32) . In short, neutrophils (2 10/ml in incubation medium) were prewarmed at 37 °C for 10 min. Subsequently, the cells were incubated with 0.25 µM 1,2,3-dihydrorhodamine (Molecular Probes) and 2 mM NaN for 5 min. The cells were incubated with the appropriate antibodies, washed, and resuspended in warm incubation medium, and were incubated with cross-linking agents for 30 min. The reactions were stopped by addition of a 30-fold excess of ice-cold PBS containing 1% (v/v) bovine serum albumin and the samples were kept on ice. Cell-associated fluorescence was measured by flow cytometry (FACScan, Becton Dickinson).

Statistical Analysis

For statistical analysis paired Student t tests were performed. p values exceeding 0.05 were not considered significant.


RESULTS

The Simultaneous Homotypic Response via FcRIIa and FcRIIIb Shows an Incomplete Summation of the Individual Responses

We first investigated the Ca response of human neutrophils after simultaneous, but specific, homotypic cross-linking of both FcR receptors (further referred to as the ``simultaneous homotypic FcR response'') and compared this response to the responses after cross-linking of each receptor alone. For this purpose, neutrophils were incubated with mAb IV.3 (anti-FcRII) and biotinylated 3G8 F(ab`) (anti-FcRIII). Neither mAb IV.3 nor 3G8 F(ab`)-biotin alone elicited an increase in [Ca](data not shown). Subsequently, GAM F(ab`) anti-Fc domains (to specifically cross-link FcRIIa) and streptavidin (to specifically cross-link FcRIIIb) were added simultaneously, which resulted in a significant rise in [Ca](Fig. 1 C and ). Neither GAM F(ab`) nor streptavidin alone induced a Ca response. Reference responses were determined by multivalent homotypic cross-linking of both receptors alone with the same agents (Fig. 1, A and B, and ). The simultaneous homotypic FcR response was significantly lower than the sum of the separate FcRIIa and FcRIIIb responses: the peak increase in [Ca]was only 79 ± 5% (mean ± S.E., n = 4) of this latter value ( p < 0.025).


Figure 1: Changes in intracellular free Ca after homotypic cross-linking of FcRIIa and FcRIIIb on human neutrophils. Indo-1-loaded neutrophils were preincubated for 5 min at 37 °C with: A, intact IV.3 mAb (10 µg/ml); B, biotinylated anti-3G8 F(ab`) (10 µg/ml); or C, with both mAb. The cells were then washed and transferred to a cuvette. Cross-linking ( arrow) was performed with: A, GAM F(ab`) anti-Fc domains (15 µg/ml); B, streptavidin (10 µg/ml); or C, with both cross-linkers together. Each curve is representative for four independent experiments.



For this inhibition to occur, ligation of one FcR, without multivalent cross-linking was not sufficient. Neither the response after FcRIIa cross-linking (the mean of the Ca increase ± S.E.: 288 nM ± 61, n = 3), nor the response after FcRIIIb cross-linking (237 nM ± 70, n = 7) was significantly changed by ligation of FcRIIIb with 3G8 Fab (293 nM ± 70, n = 3) or FcRIIa with IV.3 Fab (272 nM ± 72 n = 7), respectively. The results depicted in Fig. 1 and might suggest some competition between the signal transduction pathways used in activation via homotypic cross-linking of both FcR.

Synergistic Response Evoked by Heterotypic Cross-linking of FcRIIa and FcRIIIb

To mimic cross-linking of FcRs by immune complexes, neutrophils were incubated with anti-FcRII Fab fragments and anti-FcRIII Fab fragments, and then GAM F(ab`) was added. After cross-linking with GAM F(ab`), which binds to both anti-FcRII Fab and anti-FcRIII Fab, a pronounced increase in [Ca]was observed ( Fig. 2 and ). Neither anti-FcRII Fab nor anti-FcRIII Fab alone, nor both Fabs in combination, nor GAM F(ab`) alone induced a response (data not shown). The GAM F(ab`) concentration chosen induced a level of activation of the separate Fc receptors responses that was comparable or even lower than these responses seen with the other reagents used for cross-linking ( cf. Tables I and II).


Figure 2: Changes in intracellular free Ca after combined homotypic and heterotypic cross-linking of FcRIIa and FcRIIIb on human neutrophils. Indo-1-loaded neutrophils were preincubated for 5 min at 37 °C with: A, IV.3 Fab (10 µg/ml); B, 3G8 Fab (10 µg/ml); or C, with both mAb. The cells were then washed and transferred to a stirred cuvette. Cross-linking ( arrow) was performed with GAM F(ab`) (15 µg/ml). Curves are representative for five independent experiments.



In the experiments depicted in Fig. 2(further referred to as the ``combined FcR response''), a combination of homotypic FcR cross-linking with a substantial proportion of heterotypic cross-linking of FcRIIa and FcRIIIb may be expected. Immunofluorescence microscopy showed that, indeed, upon cross-linking of anti-FcRII Fab (FITC-labeled) and anti-FcRIII Fab (tetramethylrhodamine isothiocyanate-labeled) with GAM F(ab`), localization of FcRIIa and FcRIIIb in the same clusters on the cell surface was induced (data not shown). The magnitude of the contribution of homotypic cross-linking to the combined FcR response was determined by means of separate cross-linking of both FcR with the same agents ( Fig. 2and ). The increase in [Ca]of the combined FcR response reached a much higher level than the sum of the Ca responses initiated by FcRIIa cross-linking alone and FcRIIIb cross-linking alone. The ratio of the ``combined'' response to the sum of the separate responses was 3.03 ± 0.71 (mean ± S.E., n = 5) at antibody concentrations of 10 µg/ml and 1.62 ± 0.14 (mean ± S.E., n = 3) at antibody concentrations of 2.5 µg/ml. Thus, in the combined FcR response, in which besides homotypic cross-linking also heterotypic cross-linking can occur, a significant synergistic increase in [Ca]is observed ( p < 0.05 for both antibody concentrations). Comparison of the results depicted in Figs. 1 and 2 shows that the kinetics of the increase in [Ca]are dependent on the agent used for cross-linking the FcRs. Nevertheless, these results suggest that a synergistic increase in [Ca]is evoked after heterotypic FcRIIa FcRIIIb cross-linking.

To further explore the effect of cross-linking of FcRIIa and FcRIIIb together, the effect of stimulation with bsAb, directed against both FcRIIa and FcRIIIb, was investigated. The bsAb FcRIIxFcRIII was able to inhibit binding of both IV.3-FITC and, to a lesser extent, 3G8 F(ab`)-FITC to human neutrophils (I). To ensure the presence of the Fab recognizing CD16, binding of the bsAb to CHO cells expressing human FcRIIIb was studied (I). Although these cells did not express FcRII, a clear binding of the bsAb was observed. Hence, the bsAb consisted of IV.3 Fab and 3G8 Fab with intact antigen-binding sites.

Direct heterotypic cross-linking of one FcRIIa to one FcRIIIb by addition of the bsAb to neutrophils did not initiate an increase in [Ca](). Additional cross-linking of the bsAb with GAM F(ab`) was required to elicit a [Ca]response (further referred to as the ``bsAb response'') ( Fig. 3and ). This response was greater than the sum of the quantitative comparable separate responses obtained with comparable amounts of Fab fragments ( p < 0.005).


Figure 3: Changes in intracellular free Ca after cross-linking of bsAb FcRIIxFcRIII. Indo-1-loaded neutrophils were preincubated for 5 min at 37 °C with: A, bsAb FcRIIxFcRIII (10 µg/ml); B, bsAb FcRIIxFcRIII (5 µg/ml); C, IV.3 Fab and 3G8 Fab (both at 5 µg/ml); or D, IV.3 Fab and 3G8 Fab (both at 2.5 µg/ml). The cells were then washed and transferred to a cuvette. Cross-linking ( arrow) was performed with GAM F(ab`) at a concentration of 15 µg/ml ( A and C) or 8 µg/ml ( B and D). Curves A and C are representative for six independent experiments, curves B and D are representative for three independent experiments.



Comparison of the bsAb response (obtained with 10 µg of bsAb/ml) with the combined FcR response with the same amounts of Fab fragments (5 µg/ml of each Fab fragment) showed that the peak value of the bsAb response was consistently higher ( Fig. 3and ): an increase of 32 ± 12% (mean ± S.E., n = 6) was observed ( p < 0.05). However, the bsAb response occurred more rapidly as compared to the combined FcR response, but also appeared to be more transient. Apparently, the formation of FcRIIxFcRIII complexes by the bsAb prior to cross-linking does have an influence on the characteristics of the final response.

The Synergistic CaResponse Is Mainly Derived from Extracellular Ca

To investigate the source of the Ca in the various responses described above, changes in indo-1 fluorescence were investigated in the presence of EGTA (to prevent Ca influx) or Mn (to indirectly measure Ca influx). The peak levels of [Ca]reached under the conditions previously designated as the simultaneous homotypic FcR response and the combined FcR response were lowered to a mean level of 240 and 234 nM Ca, respectively, in the presence of EGTA, indicating that extracellular Ca contributes profoundly to the peak values reached ( Fig. 4 and ). The separate responses via FcRIIa and FcRIIIb were only slightly decreased in the presence of EGTA (). Furthermore, in neutrophils loaded with the intracellular Ca chelator, BAPTA-AM, especially the response to combined heterotypic and homotypic cross-linking was not completely abrogated (Fig. 4). This might be explained by a substantial contribution of extracellular Ca to this response overriding the buffer capacity of the BAPTA-loaded cells. In contrast, the response after cross-linking of FcRII or FcRIII alone was completely abrogated in BAPTA-loaded cells (data not shown).


Figure 4: Effect of Ca chelation on changes in [Ca] induced by FcR cross-linking. The upper figure depicts ( A) the simultaneous homotypic FcR response and ( B) the combined FcR response without chelation of Ca, the middle figure depicts these responses with chelation of extracellular Ca by EGTA and the lower figure depicts these responses with chelation of intracellular Ca by BAPTA. Loading with indo-1 and BAPTA was performed as described under ``Materials and Methods.'' Subsequently, the neutrophils were preincubated with antibodies ( A) exactly as described in the legend to Fig. 1 or ( B) exactly as described in the legend to Fig. 2. Cross-linking ( arrow) was performed with: A, streptavidin (10 µg/ml) and GAM F(ab`) anti-Fc domains (15 µg/ml); or B, GAM F(ab`) (15 µg/ml) in the presence of extracellular Ca ( upper and lower figure) or without extracellular Ca in the presence of 1 mM EGTA ( middle figure). Curves shown are representative for three to five independent experiments.



More direct evidence for an effect of heterotypic FcR cross-linking on influx of extracellular Ca was obtained by Mn quenching experiments, in which extracellular Mn enters the cells via Ca channels (25) . In our experiments, an emission wavelength of 446 nm was used, which made the fluorescence of indo-1-loaded neutrophils insensitive for changes in [Ca](data not shown). Combined homotypic and heterotypic FcR cross-linking, induced after preincubation with both FcR antibodies together or with the bsAb, resulted in a sharp decrease in indo-1 fluorescence, i.e. a high rate of Mn influx (Fig. 5, A and B). Under conditions of simultaneous homotypic cross-linking of both FcR, the rate of Mn influx was much lower (Fig. 5 C). After cross-linking of FcRII or FcRIII alone (Fig. 5, D and E), Mn influx appeared to be only slightly higher than in unstimulated cells (Fig. 5 F). The enhancement of Mn influx was not observed upon co-cross-linking of FcRIIa and MHC class I or of FcRIIIb with MHC class I (data not shown).


Figure 5: Effect of FcR cross-linking on Ca influx as measured by Mn-dependent quenching of indo-1 fluorescence. Indo-1 emission was measured at the Ca-independent wavelength of 446 nm as explained in the text. The indo-1-loaded neutrophils were preincubated for 5 min at 37 °C as indicated below, washed, resuspended in incubation medium with 0.2 µM CaCl, and transferred to a cuvette. Subsequently, 0.5 mM MnCl was added ( first arrow) and cross-linking was performed ( second arrow). To completely quench indo-1 fluorescence with Mn, digitonin (10 µM) was added ( third arrow). A, combined FcR response: preincubation with IV.3 Fab (10 µg/ml) + 3G8 Fab (10 µg/ml), cross-linking with GAM F(ab`) (15 µg/ml). B, bsAb response: preincubation with bsAb (10 µg/ml), cross-linking with GAM F(ab`) (15 µg/ml). C, simultaneous homotypic FcR response: preincubation with IV.3 (10 µg/ml) + 3G8 F(ab`)-biotin (10 µg/ml), cross-linking with GAM F(ab`) anti-Fc domains (15 µg/ml) + streptavidin (10 µg/ml). D, FcRII response: preincubation with IV.3 Fab (10 µg/ml), cross-linking with GAM F(ab`) (15 µg/ml). E, FcRIII response: preincubation with 3G8 Fab (10 µg/ml), cross-linking with GAM F(ab`) (15 µg/ml). F, preincubation without FcR antibodies, cross-linking with GAM F(ab`) (15 µg/ml). Curves are representative for two to five independent experiments.



Heterotypic Clustering of FcR Induces a Higher Depletion of Intracellular CaStores

The results depicted above indicated that especially upon heterotypic cross-linking of FcRIIa and FcRIIIb, Ca influx from the extracellular environment is induced. In several cell types (including neutrophils), the depletion of intracellular stores can activate Ca influx (30, 33) . To assess the depletion of Ca stores under the various conditions of FcR cross-linking, stimulations were performed in the presence of EGTA. After 3 min stimulation, ionomycin was added to mobilize all Ca that had remained in the stores (). Especially after heterotypic cross-linking of FcR the response to ionomycin was very poor, indicating that under these conditions depletion of Ca stores had occurred to a much higher degree than after homotypic simultaneous cross-linking or the separate FcR cross-linking. Hence, an increased store depletion might cause the Ca influx observed after heterotypic FcR cross-linking.

Immune Complexes Also Induce CaInflux Characteristic for the Synergistic Response

The ability of immune complexes to bind to both FcRIIa and FcRIIIb renders it likely that the synergism we observed is relevant for physiological FcR stimulation. To test this hypothesis, neutrophils were stimulated by heat-aggregated IgG. The [Ca]increase induced by heat-aggregated IgG could be partially blocked by either anti-FcRII Fab or anti-FcRIII Fab (data not shown), indicating involvement of both Fc receptors in this response. Moreover, heat-aggregated IgG induced a significant Mn influx (Fig. 6) that was inhibited by either anti-FcRII Fab or anti-FcRIII Fab (Fig. 6), indicating a role for both Fc receptors in inducing this response. This conclusion was further supported by the observation that neutrophils from an FcRIII-negative donor showed hardly any Ca influx upon stimulation with heat-aggregated IgG as compared to control neutrophils (Fig. 7), also indicating a contribution of the FcRIIIb in this process.


Figure 6: Effect of heat-aggregated IgG on Ca influx as measured by Mn-dependent quenching of indo-1 fluorescence. Indo-1 emission was measured at the Ca-independent wavelength of 446 nm as explained in the text. Neutrophils were diluted five times in incubation medium without Ca before being transferred to the cuvette. Subsequently, 0.5 mM MnCl was added ( first arrow) and stimulation with heat-aggregated IgG (1 mg/ml) was performed ( second arrow) with preincubation of 3G8 Fab (10 µg/ml) ( A) or IV.3 Fab (10 µg/ml) ( B) or without preincubation ( C). To completely quench indo-1 fluorescence with Mn, digitonin (10 µM) was added ( third arrow). Curves are representative for two independent experiments.




Figure 7: Effect of heat-aggregated IgG on Ca influx in FcRIIIb-nega-tive neutrophils as measured by Mn-dependent quenching of indo-1 fluorescence. Indo-1 emission was measured at the Ca-independent wavelength of 446 nm as explained in the text. Neutrophils were diluted five times in incubation medium without Ca before being transferred to the cuvette. Subsequently, 0.5 mM MnCl was added ( first arrow) and stimulation with heat-aggregated IgG (1 mg/ml) was performed ( second arrow) in FcRIIIb-negative neutrophils ( A) or control neutrophils ( B). The response of the control neutrophils was representative for the neutrophils of five different donors. To completely quench indo-1 fluorescence with Mn, digitonin (10 µM) was added ( third arrow).



Heterotypic Cross-linking of FcRIIa and FcRIIIb Induces a Synergistic Activation of the Respiratory Burst

To investigate whether heterotypic cross-linking of FcRIIa and FcRIIIb affects other neutrophil responses induced by FcR ligation, activation of the respiratory burst was measured. Neither anti-FcRII Fab, anti-FcRIII Fab, nor GAM F(ab`) alone induced an increase in respiratory burst activity. The homotypic cross-linking of both FcRIIa and FcRIIIb induced some activation as shown before by Hundt et al.(4) . The combined FcR response was significantly higher then the sum of the homotypic FcR responses ( p < 0.0125) (Fig. 8). The increase in the respiratory burst activity of the combined FcR response was 2.04 ± 0.3 times higher than the sum of the separate responses, indicating a synergistic increase in respiratory burst activity upon heterotypic cross-linking of both Fc receptors on human neutrophils.


Figure 8: Activation of the respiratory burst after heterotypic cross-linking of FcRIIa and FcRIIIb on human neutrophils. Neutrophils were loaded with 0.25 µM 1,2,3-dihydrorhodamine ( DHR). The cells were pretreated with IV.3 Fab (5 µg/ml) or 3G8 Fab (5 µg/ml) or both IV.3 Fab and 3G8 Fab for 5 min at 37 °C, washed, and incubated with GAM F(ab`) (15 µg/ml) for 30 min, as indicated. As control, no antibodies were added. For comparison neutrophils were treated for 2 min with 1 µM platelet-activating factor ( PAF) and for 10 min with 1 µM formyl-methionyl-leucine-phenylalanine ( fMLP). Fluorescence of DHR was measured by flow cytometry. Values were given as mean fluorescence ± S.E. of four independent experiments.




DISCUSSION

Recent studies have established that both FcRs on human neutrophils can transduce signals (4, 12, 15) . In the present study we have investigated the effects of possible interactions between signaling via FcRIIa and FcRIIIb. Changes in [Ca]were used as indicator for signal transduction events. In considering possible interactions between signal transduction via FcRIIa and FcRIIIb, one should differentiate at least three situations. First, a mono- or divalent ligation of one receptor, without triggering a signal, might have a modulating effect on a specific stimulation via the other receptor. Second, the signal transduction pathway activated by homotypic cross-linking of one receptor might interact with the pathway simultaneously triggered by homotypic cross-linking of the other receptor. Third, heterotypic clusters of FcRIIa and FcRIIIb might initiate a type of signal transduction that has quantitative or qualitative properties distinct from that of homotypic clusters.

Our results show that ligation of one receptor did not induce a modulation of the Ca response subsequently elicited via the other receptor. This is in contrast to the proposal of some investigators (20, 21) of modulation of the Ca response elicited via FcRIIIb by monovalent ligation of FcRIIa. In these studies aggregated IgG (20) or insoluble immune complexes (21) were used as stimuli. Although these complexes were proposed to bind specifically to FcRIIIb, a simultaneous binding to FcRIIa is difficult to exclude due to the much lower expression of this receptor. This could account for the observed inhibition by ligation of FcRII.

It has been suggested that cross-linking of both FcRs is required to achieve a full response of the neutrophil to immune complexes (21) . Indications for a synergistic effect of simultaneous activation via both FcRs have been found in some studies (4, 34) . However, the level at which this synergism takes place has not been revealed.

Our results show that a synergistic effect by dual receptor activation is only observed when cross-linking of FcRIIa to FcRIIIb is possible ( Fig. 2 and ). Simultaneous stimulation via both receptors performed in a way that allowed only homotypic cross-linking of both receptors to occur did not result in synergism, and even had an inhibiting effect. Under those circumstances, the response did not reach the sum of the separate homotypic responses ( Fig. 1and ). Additional evidence for the requirement of heterotypic cross-linking of FcRIIa and FcRIIIb for the generation of a synergistic [Ca]response was obtained from experiments with the bsAb FcRIIxFcRIII. The heterotypic cross-linking by bsAb also induced a Ca response much higher than obtained after separate FcR cross-linking ( Fig. 3and ). The observation that immune complexes induced hardly any Ca influx in FcRIIIb-negative neutrophils, in contrast to normal neutrophils, also indicated an important contribution of FcRIIIb in the immune complex-induced Ca influx (Fig. 7).

Taken together, the interaction of FcRIIa with FcRIIIb in heterotypic clusters leads to a response that is quantitatively distinct from the result obtained after homotypic cross-linking. Whether this synergistic effect of heterotypic cross-linking is only a quantitative phenomenon, induced by more extensive cluster formation, or also reflects qualitative changes in the signal transduction elicited by heterotypic clusters, remains to be elucidated. However, we did observe that under conditions of heterotypic cross-linking, the influx of extracellular Ca was especially increased. Experiments in the presence of EGTA indicated that the individual FcRIIa and FcRIIIb responses exhibit a slight dependence on Ca influx from the extracellular medium ( Fig. 4and ). With increasing responses we observed an enhanced Ca influx from the extracellular medium, especially under conditions of heterotypic cross-linking of FcR (Fig. 5). This enhanced Ca influx is probably caused by enhanced store depletion, according to the model of Putney (33) . Our results show a higher degree of intracellular Ca mobilization upon heterotypic clustering as compared to simultaneous homotypic cross-linking (). Even with much higher concentrations of cross-linking agents (up to 50 µg/ml streptavidin or Fc-specific GAM F(ab`) instead of 10 and 15 µg/ml, respectively, as used in most experiments) in the simultaneous homotypic response the level of store depletion was always lower than for the heterotypic FcR response (data not shown).

During phagocytosis, accumulation of Ca stores in the area around the phagosome has been described supporting locally the induction of high Ca concentrations (35) . Possibly, accumulation of Ca stores to the site of cross-linked receptors under conditions of heterotypic cross-linking contributes to the response. Alternatively, the formation of heterotypic clusters of Fc receptors might allow additional signals by trans-phosphorylations of associated proteins or FcRII itself that are instrumental for the synergistic response to occur (36) .

The heterotypic cross-linking of FcRIIa and FcRIIIb also induced synergism in functional responses of neutrophils. Synergism in FcR-mediated phagocytosis has recently been described in literature (37) . We observed also synergistic activation of the respiratory burst (Fig. 8). This seems in contrast to earlier studies of our group, showing the oxidative burst to be normal in neutrophils from paroxysmal nocturnal hemoglobinuria patients when stimulated with immune complexes (2) . Blood cells from paroxysmal nocturnal hemoglobinuria patients lack glycosylphosphatidylinositol-anchored proteins, such as FcRIIIb on neutrophils. However, neutrophils from paroxysmal nocturnal hemoglobinuria patients still express approximately 10% of normal levels of FcRIIIb (2) , which is still a reasonable number as compared to the number of FcRIIa on the neutrophils. This amount of FcRIIIb may still play a role in the heterotypic interaction between Fc receptors in neutrophils.

In conclusion, our study shows that formation of heterotypic clusters of FcR on human neutrophils greatly enhances Ca responses by an effect on Ca influx and induces a synergistic activation of the respiratory burst. In this way, heterotypic cluster formation of FcR may be a requirement for full generation of effector functions (4, 34) . The ability of immune complexes to evoke a Ca influx suggests that this synergism is physiologically relevant.

  
Table: Peak increases in [Ca]after homotypic cross-linking of FcR on human neutrophils

Peak increases above resting values under the various conditions of stimulation were calculated. Experimental details are given in the legend to Fig. 1. Results are the mean ± S.E. of four independent experiments.


  
Table: Synergism of the combined FcR response

The results of different experiments on the combined FcR response are shown together with the reference FcR responses determined in the same experiments. The experiments were carried out as described in the legend to Fig. 2 with antibody concentrations as indicated in the table. Results given are the mean ± S.E. of the number of experiments indicated between parentheses.


  
Table: Characterization of BsAb FcRIIxFcRIII

Fixed neutrophils, FcRIIIb-transfected CHO cells (CHO cells) or wild type CHO cells (CHO cells) were incubated with BsAb FcRIIxFcRIII, aspecific mouse IgG1, IV.3 Fab, or 3G8 F(ab`) as described under ``Materials and Methods.'' After washing, the cells were incubated with GAM-FITC (62.5 µg/ml), IV.3-FTTC (2.5 µg/ml), or 3G8 F(ab`)-FITC (2.5 µg/ml) for 30 min at 4 °C. After another washing step, fluorescence data were collected from 5000 cells by flow cytometry. Results are the mean of two independent experiments.


  
Table: BsAb response compared to the combined FcR response

The results of different experiments are shown in which the BsAb response and the combined FcR response were compared. The experiments were performed as described in the legends to Figs. 3 and 4. Peak increases above resting values under the various conditions of stimulation were calculated. Results given are the mean ± S.E. of the number of experiments indicated.


  
Table: Effect of extracellular Ca on changes in [Ca]induced by FcR cross-linking

The simultaneous homotypic FcR response, the combined FcR response, and the separate FcR responses were measured in the presence of 1 mM extracellular Ca or in the presence of EGTA as described under ``Materials and Methods.'' The cells were pretreated with IV.3, 3G8 F(ab`)-biotin, IV.3 Fab, or 3G8 Fab (each 10 µg/ml) or a combination of these antibodies for 5 min at 37 °C, were washed and were incubated with streptavidine (10 µg/ml) or GAM F(ab`) (15 µg/ml), as indicated. Peak increases above resting values under the various conditions of stimulation were calculated. Results given are the mean ± S.E. of the number of experiments indicated.


  
Table: Effect of FcR cross-linking on the depletion of intracellular Ca stores

The simultaneous homotypic FcR response, the combined FcR response, and the separate FcR responses were performed in the presence of 1 mM EGTA as described under ``Materials and Methods.'' After 3 min 1 µM ionomycin was added to quantitate the amount of Ca still present in the stores. Concentrations of cross-linking agents were the same as separate responses. Results given are the mean ± S.E. of the number of experiments indicated.



FOOTNOTES

*
This work was supported by Grant 900-512-092 from the Netherlands Organization for Scientific Research (NWO). 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: c/o Publication Secretariat, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands. Tel.: 31-20-5123317; Fax: 31-20-5123310.

The abbreviations used are: FcR, the receptors for IgG; GAM, goat anti-mouse immunoglobulin antibodies; mAb, mouse monoclonal antibodies; bsAb, bispecific antibodies; PAGE, polyacrylamide gel electrophoresis; [Ca], intracellular free Ca concentration; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; CHO, Chinese hamster ovary; SPDP, N-succinimidyl-3-(2-pyridyldithiol)propionate; BAPTA, 1,2-bis-( O-aminophenoxyl)ethane- N,N,N`,N`-tetraacetic acid; MHC, major histocompatibility complex.


ACKNOWLEDGEMENTS

We thank Lily Kannegieter for purification of the bispecific antibody, Martin de Boer for technical assistance with photography, and Masja de Haas for critically reading the manuscript.


REFERENCES
  1. Huizinga, T. W. J., Kerst, M., Nuyens, J. H., Vlug, A., Von dem Borne, A. E. G. Kr., Roos, D., and Tetteroo, P. A. T. (1989) J. Immunol. 142, 2359-2364 [Abstract/Free Full Text]
  2. Huizinga, T. W. J., Van der Schoot, C. E., Jost, C., Klaassen, R., Kleijer, M., Von dem Borne, A. E. G. Kr., Roos, D., and Tetteroo, P. A. T. (1988) Nature 333, 667-669 [CrossRef][Medline] [Order article via Infotrieve]
  3. Huizinga, T. W. J., Van Kemenade, F., Koenderman, L., Dolman, K. M., Von dem Borne, A. E. G. Kr., Tetteroo, P. A. T., and Roos, D. (1989) J. Immunol. 142, 2365-2369 [Abstract/Free Full Text]
  4. Hundt, M., and Schmidt, R. E. (1992) Eur. J. Immunol. 22, 811-816 [Medline] [Order article via Infotrieve]
  5. Anderson, C. L., Shen, L., Eicher, D. M., Wewers, M. D., and Gill, J. K. (1990) J. Exp. Med. 171, 1333-1345 [Abstract]
  6. Willis, H. E., Browder, B., Feister, A. J., Mohanakumar, T., and Ruddy, S. (1988) J. Immunol. 140, 234-239 [Abstract/Free Full Text]
  7. Indik, Z., Kelly, C., Chien, P., Levinson, A. I., and Schreiber, A. D. (1991) J. Clin. Invest. 88, 1766-1771 [Medline] [Order article via Infotrieve]
  8. Graziano, R. F., and Fanger, M. W. (1987) J. Immunol. 139, 3536-3541 [Abstract/Free Full Text]
  9. Rosales, C., and Brown, E. J. (1991) J.Immunol. 146, 3937-3944 [Abstract/Free Full Text]
  10. Lanier, L. L., Ruitenberg, J. J., and Phillips, J. H. (1988) J. Immunol. 141, 3478-3485 [Abstract/Free Full Text]
  11. Tosi, M. F., and Berger, M. (1988) J. Immunol. 141, 2097-2103 [Abstract/Free Full Text]
  12. Kimberly, R. P., Ahlstrom, J. W., Click, M. E., and Edberg, J. C. (1990) J. Exp. Med. 171, 1239-1255 [Abstract/Free Full Text]
  13. Salmon, J. E., Brogle, N. L., Edberg, J. C., and Kimberly, R. P. (1991) J. Immunol. 146, 997-1004 [Abstract/Free Full Text]
  14. Shen, L., Graziano, R. F., and Fanger, M. W. (1989) Mol. Immunol. 26, 959-969 [Medline] [Order article via Infotrieve]
  15. Huizinga, T. W. J., Dolman, K. M., Van der Linden, N. J. M., Kleijer, M., Nuijens, J. H., Von dem Borne, A. E. G. Kr., and Roos, D. (1990) J. Immunol. 144, 1432-1437 [Abstract/Free Full Text]
  16. Salmon, J. E., Kapur, S., and Kimberly, R. P. (1987) J. Exp. Med. 166, 1798-1813 [Abstract]
  17. Crockett-Torabi, E., and Fantone, J. C. (1990) J. Immunol. 145, 3026-3032 [Abstract/Free Full Text]
  18. Walker, B. A. M., Hagenlocker, B. E., Stubbs, E. B., Sandborg, R. R., Agranoff, B. W., and Ward, P. A. (1991) J. Immunol. 146, 735-741 [Abstract/Free Full Text]
  19. Boros, P., Odin, J. A., Muryoi, T., Masur, S. K., Bona, C., Unkeless, J. C. (1991) J. Exp. Med. 173, 1473-1482 [Abstract]
  20. Naziruddin, B., Duffy, B. F., Tucker, J., and Mohanakumar, T. (1992) J. Immunol. 149, 3702-3709 [Abstract/Free Full Text]
  21. Brunkhorst, B. A., Strohmeier, G., Lazzari, K., Weil, G., Melnick, D., Fleit, H. B., and Simons, E. R. (1992) J. Biol. Chem. 267, 20659-20666 [Abstract/Free Full Text]
  22. Huizinga, T. W. J., Kuypers, R. W. A. M., Kleijer, M., Schulpen, T. W. J., Cuypers, H. T. M., Roos, D., and Von dem Borne, A. E. G. Kr. (1990) Blood 76, 1927-1932 [Abstract]
  23. Fleit, H. B., Wright, S. D., and Unkeless, J. C. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3275-3279 [Abstract]
  24. Rosenfeld, S. I., Looney, R. J., Leddy, J. P., Phipps, D. C., Abraham, G. N., and Anderson, C. L. (1985) J. Clin. Invest. 76, 2317-2322 [Medline] [Order article via Infotrieve]
  25. Ory, P. A., Clark, M. R., Talhouk, A. S., and Goldstein, I. M. (1991) Blood 77, 2682-2687 [Abstract]
  26. Koenderman, L., Yazdanbakhsh, M., Roos, D., and Verhoeven, A. J. (1989) J. Immunol. 142, 623-628 [Abstract/Free Full Text]
  27. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450 [Abstract]
  28. Bijsterbosch, M. K., Rigley, K. P., and Klaus, G. G. B. (1986) Biochem. Biophys. Res. Commun. 137, 500-506 [Medline] [Order article via Infotrieve]
  29. Hallam, T. J., Jacob, R., and Merritt, J. E. (1988) Biochem. J. 255, 179-184 [Medline] [Order article via Infotrieve]
  30. Demaurex, N., Monod, A., Lew, D. P., and Krause, K.-H. (1994) Biochem. J. 297, 595-601 [Medline] [Order article via Infotrieve]
  31. Emmendörffer, A., Hecht, M., Lohman-Matthes, M.-L., and Roesler, J. (1990) J. Immunol. Methods 131, 269-275 [CrossRef][Medline] [Order article via Infotrieve]
  32. Rothe, G., Emmendörffer, A., Oser, A., Roesler, J., and Valet, G. (1991) J. Immunol. Methods 138, 133-135 [CrossRef][Medline] [Order article via Infotrieve]
  33. Putney, J. W., Jr. (1990) Cell Calcium 11, 611-624 [Medline] [Order article via Infotrieve]
  34. Edberg, J. C., Salmon, J. E., and Kimberly, R. P. (1992) Immunol. Res. 11, 239-251 [Medline] [Order article via Infotrieve]
  35. Stendahl, O., Krause, K.-H., Krischer, J., Jerström, P., Theler, J.-M., Clark, R. A., Carpentier, J.-L., and Lew, D. P. (1994) Science 265, 1439-1441 [Medline] [Order article via Infotrieve]
  36. Zhou, M., and Brown, E. J. (1994) J. Cell Biol. 125, 1407-1416 [Abstract]
  37. Edberg, J. C., and Kimberly, R. P. (1994) J. Immunol. 152, 5826-5835 [Abstract/Free Full Text]

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