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The Journal of Immunology, 2007, 179: 2918-2924.
Copyright © 2007 by The American Association of Immunologists, Inc.
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FcR {gamma}-Chain Dependent Signaling in Immature Neutrophils Is Mediated by Fc{alpha}RI, but Not by Fc{gamma}RI1

Marielle A. Otten*,{dagger}, Jeanette H. W. Leusen*, Esther Rudolph*,{dagger}, Joke A. van der Linden*, Robert H. J. Beelen{dagger}, Jan G. J. van de Winkel*,{ddagger} and Marjolein van Egmond2,{dagger},§

* Immunotherapy Laboratory, Department of Immunology, University Medical Center, Utrecht, The Netherlands; {dagger} Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands; {ddagger} Genmab, Utrecht, The Netherlands; and § Department of Surgical Oncology, VU University Medical Center, Amsterdam, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Neutrophil-mediated tumor cell lysis is more efficiently triggered by Fc{alpha}RI (CD89), than by Fc{gamma}RI (CD64). This difference is most evident in immature neutrophils in which Fc{gamma}RI-mediated tumor cell lysis is absent. In this study, we show that FcR {gamma}-chain-dependent functions (such as Ab-dependent cellular cytotoxicity and respiratory burst), as well as signaling (calcium mobilization and MAPK phosphorylation), were potently triggered via Fc{alpha}RI, but not via Fc{gamma}RI, in immature neutrophils. Internalization, an FcR {gamma}-chain-independent function, was, however, effectively initiated via both receptors. These data suggest an impaired functional association between Fc{gamma}RI and the FcR {gamma}-chain, which prompted us to perform coimmunoprecipitation experiments. As a weaker association was observed between Fc{gamma}RI and FcR {gamma}-chain, compared with Fc{alpha}RI and FcR {gamma}-chain, our data support that differences between Fc{alpha}RI- and Fc{gamma}RI-mediated functions are attributable to dissimilarities in association with the FcR {gamma}-chain.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immune cell signaling can be initiated by binding of Abs to FcRs. After activation, FcRs trigger effector functions such as Ab-dependent cellular cytotoxicity (ADCC),3 release of oxygen radicals, endocytosis, phagocytosis, degranulation, Ag presentation, and release of inflammatory mediators (for review, see Ref. 1). In humans, one class of IgA FcR (Fc{alpha}RI, CD89) and three classes of leukocyte IgG FcRs, i.e., Fc{gamma}RI (CD64), Fc{gamma}RII (CD32), and Fc{gamma}RIII (CD16), have been described (2, 3). Fc{gamma}RIIIb is a GPI-linked protein (4). Fc{gamma}RIIa bears a so-called activatory ITAM signaling motif in its cytoplasmic tail, which is required for induction of effector functions, whereas Fc{gamma}RIIb harbors an inhibitory ITIM-signaling motif (5, 6). The {alpha}-chains of Fc{alpha}RI, Fc{gamma}RI, and Fc{gamma}RIIIa lack such signaling motifs. Therefore, these latter receptors associate with the common ITAM-containing FcR {gamma}-chain to mediate effector functions (7, 8).

Of all Fc{gamma}R, cell distribution of Fc{gamma}RI most closely resembles Fc{alpha}RI expression. Both receptors are selectively expressed on cells of the myeloid lineage, including monocytes, macrophages, and dendritic cells (2, 9). Furthermore, neutrophils constitutively express Fc{alpha}RI, whereas Fc{gamma}RI expression can be induced on these cells by addition of either IFN-{gamma} or G-CSF (10, 11). Neutrophils represent the most populous cytotoxic effector cell subset within the blood; neutrophil numbers can be increased by treatment with G-CSF (12). They play a prominent role in bacterial infections (13), but exert well-documented antitumor properties as well because neutrophils have been shown to play a role in tumor rejection in the presence of antitumor Ab, both in vitro and in vivo (14, 15, 16, 17). Therefore, we—and others—studied the potential of Fc{alpha}RI (18, 19) and Fc{gamma}R (20, 21) on these immune cells to induce tumor cell lysis.

Fc{gamma}RI was reported to represent the most potent neutrophil Fc{gamma}R for induction of ADCC (20, 21). However, compared with Fc{gamma}RI, tumor cell lysis was more efficient via targeting of neutrophil Fc{alpha}RI (18, 19). This difference was most evident in bone marrow neutrophils, in which Fc{gamma}RI-initiated tumor cell killing was hampered (19). Interestingly, cell surface expression of either receptor depends on association with the common FcR {gamma}-chain, which was shown in Fc{alpha}RI- and Fc{gamma}RI-transgenic mice, in which surface expression was lost when these transgenic mice were crossed with FcR {gamma}-chain-deficient mice (22, 23). Furthermore, for initiation of most immune effector functions, both receptors depend on signaling via the FcR {gamma}-chain as well (24, 25). Therefore, in the present work, we studied the underlying mechanisms behind the discrepancies between Fc{alpha}RI- and Fc{gamma}RI-mediated effector functions in more detail.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Antibodies

Ab A77 (murine (m) IgG1 anti-Fc{alpha}RI), m22 (mIgG1 anti-Fc{gamma}RI), and 520C9 (mIgG1 anti-Her-2/neu) were produced from hybridomas (Medarex). Fc{gamma}RIxHer-2/neu bispecific Ab (BsAb) (22 x 520C9; MDX-H210) was also obtained from Medarex. Fc{alpha}RIxHer-2/neu BsAb (A77 x 520C9) was produced by chemically cross-linking F(ab') of mAb 520C9 with F(ab') of mAb A77 as described (26). For neutrophil staining, FITC-conjugated anti-CD11b mAb (Immunotech), PE-conjugated anti-CD16 mAb (BD Biosciences), and FITC-conjugated anti-human CD66b mAb (Serotec) were used. FITC-conjugated and unconjugated F(ab')2 of goat anti-mouse IgG1 mAb were purchased from Southern Biotechnology Associates. F(ab')2 of FITC-labeled rabbit anti-goat IgG (H + L) Ab were obtained from Jackson ImmunoResearch Laboratories. For Western blot analyses, rabbit anti-FcR {gamma}-chain Ab (Upstate Biotechnology), rabbit anti-phospho-p44/42 MAPK, or rabbit anti-total MAPK Ab (Cell Signaling Technology), and peroxidase (PO)-conjugated goat anti-rabbit Ab (Pierce Biotechnology) were used.

Cell lines

The breast carcinoma cell line SK-BR-3, which overexpresses the tumor-associated Ag Her-2/neu, was obtained from the American Type Culture Collection. Cells were cultured in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FCS and antibiotics (RPMI 1640/10%). SK-BR-3 cells were harvested using trypsin-EDTA (Invitrogen Life Technologies).

Blood and bone marrow donors

A peripheral blood sample (30 ml) was drawn from healthy donors receiving recombinant human G-CSF (Neupogen; 5 µg/kg body weight, twice daily for 5 days; Amgen). Bone marrow samples were obtained from cardiac patients undergoing surgery. Studies were approved by the Medical Ethical Committee of Utrecht University (Utrecht, The Netherlands), in accordance with the Declaration of Helsinki. All donors gave informed consent.

Neutrophil isolation

Neutrophils from healthy G-CSF-stimulated donors (G-CSF neutrophils) were used as positive control for Fc{gamma}RI-mediated effector functions and were isolated from heparin anticoagulated peripheral blood samples by standard Ficoll-Histopaque (Sigma-Aldrich) density gradient centrifugation.

Bone marrow neutrophils were isolated as described previously (19). In short, erythrocytes were removed by incubation for 5–10 min at 4°C with a lysis solution of pH 7.4 (0.16 M ammonium-chloride, 0.01 M potassium bicarbonate, and 0.1 mM sodium edetate), after which cells were separated by discontinuous Percoll gradient centrifugation (successively 81, 62, 55, 50, and 45% of Percoll). Percoll layers 1 and 5 in the gradient contained nonmyeloid cells, lipids, cellular debris, and remaining erythrocytes, respectively. Percoll layers 2, 3, and 4 comprised different neutrophil maturation stages.

Maturation status and purity of isolated human bone marrow cells was confirmed by cytospin preparations and staining with FITC-conjugated anti-CD11b mAb and PE-conjugated anti-CD16 mAb, as described previously (19, 27). Purity of isolated blood neutrophils was confirmed by cytospin preparation and staining with FITC-conjugated anti-human CD66b mAb. Neutrophil surface expression of Fc{alpha}RI and Fc{gamma}RI was determined with mAb A77 (Fc{alpha}RI) or m22 (Fc{gamma}RI), respectively (10 µg/ml), followed by incubation with FITC-conjugated F(ab')2 of goat anti-mouse IgG1 mAb. Cells were analyzed on a FACScan (BD Biosciences). In all experiments, neutrophil purity exceeded 95% and cell viability was >98%, as determined by trypan blue exclusion.

Ab-dependent cellular cytotoxicity

51Cr-release assays were performed as previously described (21). Briefly, SK-BR-3 target cells were incubated for 2 h at 37°C with 51Cr (100 µCi/1 x 106 cells; Amersham), washed, and plated in 96-well round-bottom microtiter plates (5 x 103 cells/well), together with 4 x 105 neutrophils/well (E:T ratio of 80:1) in the presence of 2 µg/ml BsAb. After a 4-h incubation period at 37°C, 51Cr release in the supernatant was measured as cpm. The percentage of tumor cell lysis was calculated as follows: (experimental cpm – basal cpm)/(maximal cpm – basal cpm) x 100%.

Respiratory burst assay

Neutrophils were incubated with anti-Fc{alpha}RI (A77), anti-Fc{gamma}RI (m22), or, as an isotype control, irrelevant (520C9) mAb (10 µg/ml) for 30 min at 4°C. After washing, F(ab')2 of goat anti-mouse IgG1 mAb were added to cross-link neutrophil FcRs, and tubes were placed in a 953 LB Biolumat (Berthold). Luminol (150 mM) was injected in all tubes and light emission was recorded continuously for 30 min at 37°C. Addition of fMLP to neutrophils was used as a positive control.

Calcium mobilization assay

Neutrophils were labeled with SNARF-1 (2.8 µM) and Fluo-3 (1.4 µM) (Invitrogen Life Technologies) for 30 min at 37°C. After washing, cells were incubated with anti-Fc{alpha}RI (A77) or anti-Fc{gamma}RI (m22) mAb (10 µg/ml) for 30 min at 4°C. Cells were washed twice and resuspended in calcium mobilizing buffer. FcRs were cross-linked with F(ab')2 fragments of goat anti-mouse IgG1 mAb; intracellular-free calcium levels were measured by FACScan. The first 20 s of each run, before cross-linking, were used to establish baseline intracellular calcium levels. fMLP was added to neutrophils as a positive control. To correct for variations between donors, levels of calcium flux were determined by the area under the curve (AUC), corrected for background levels by secondary Ab only.

Internalization assay

Neutrophils were preincubated with 20% pooled human serum to prevent aspecific binding to IgG FcRs (30 min at 4°C). Thereafter, cells were incubated for 30 min at 4°C with anti-Fc{alpha}RI (A77), anti-Fc{gamma}RI (m22) or, as an isotype control, irrelevant (520C9) mAb. After that, cells were washed and incubated with F(ab')2 of goat anti-mouse IgG1 mAb (30 min at 4°C). Samples were then split. One sample was kept at 4°C to measure total surface expression of the FcRs. The other sample was put at 37°C for the indicated time points to allow internalization, which was stopped by adding ice-cold FACS buffer. Remaining Fc{alpha}RI and Fc{gamma}RI surface expression was visualized by staining for 30 min at 4°C with F(ab')2 of FITC-labeled rabbit anti-goat IgG (H + L) Ab. Internalization of FcRs at 37°C was calculated as percentage of initial FcR surface expression, which was determined in 4°C samples.

MAPK phosphorylation assay

Neutrophils were incubated with anti-Fc{alpha}RI (A77) or anti-Fc{gamma}RI (m22) mAb (10 µg/ml) for 30 min at 4°C. After washing, FcRs were cross-linked with F(ab')2 of goat anti-mouse IgG1 mAb at 37°C for the indicated time points. Ice-cold PBS was added to stop reactions, after which samples were boiled in reducing Laemmli sample buffer, run on 10% SDS-PAGE gels, and electrotransferred to nitrocellulose membranes (0.45 µm; Millipore). Membranes were blocked with 5% BSA (Roche Diagnostics) and probed with rabbit anti-phospho-p44/42 MAPK, or rabbit anti-total MAPK Ab for 2 h. Following washing, membranes were incubated for an additional hour with PO-conjugated goat anti-rabbit Ab. Staining was visualized using the ECL detection system (Amersham).

Coimmunoprecipitation

Interaction of Fc{alpha}RI and Fc{gamma}RI with the FcR {gamma}-chain was measured by lysing neutrophils (5 x 107) with RIPA buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40 (NP40), 0.5% deoxycholate, and 0.1% SDS) or NP40 buffer (10 mM Tris (pH 7.4), 137 mM NaCl, 13.5 mM sodium pyrophosphate, 51.9 mM 4-fluro-3-nitrophenyl azide, 10% glycerin, 0.5% NP40), both supplemented with 1 mM PMSF, 250 µM sodium orthovanadate, 10 mM DTT, and a protease inhibitor mixture (Roche Diagnostics) for 30 min at 4°C. Homogenates were spun for 20 min at 13,000 rpm. Supernatants were precleared head over head for 30 min at 4°C with protein A/G beads (Santa Cruz Biotechnology). After that, beads were removed, and anti-Fc{alpha}RI (A77), anti-Fc{gamma}RI (m22) or, as an isotype control, irrelevant (520C9) mAb was added overnight (4°C, head over head). Protein A/G beads were added for 3 additional hours (4°C, head over head), after which beads were washed three times with lysis buffer and boiled in reducing Laemmli sample buffer. Samples were run on 15% SDS-PAGE gels, electrotransferred to nitrocellulose membranes (0.45 µm; Millipore), and membranes were blocked with 5% low-fat milk powder in PBS. Membranes were probed with rabbit anti-FcR {gamma}-chain Ab, followed by incubated with PO-conjugated goat anti-rabbit Ab. Staining was visualized using the ECL detection system (Amersham). Films were scanned with a GS-700 Imaging Densitometer and analyzed with Quantity One Software (both Bio-Rad).

Statistical analysis

Data are shown as mean ± SD. Group data are shown as mean ± SEM. Statistical differences were determined using the two-tailed unpaired Student’s t test or ANOVA. Significance was accepted when p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
FcR expression and neutrophil-mediated tumor cell killing

To study the mechanisms underlying the differences in Fc{alpha}RI and Fc{gamma}RI-mediated effector functions in neutrophil precursor subsets, cells were isolated from bone marrow and separated into three populations as described (19). In short, Percoll layer 2 (P2 neutrophil precursors) comprised the earliest neutrophil precursors, which are characterized by intermediate CD11b and low CD16 expression, as well as a round- to kidney-shaped nucleus. Percoll layer 3 (P3 neutrophil precursors) contained "intermediate" neutrophil precursors (defined by intermediate CD11b expression, heterogeneous CD16 expression, and a horseshoe-shaped nucleus). Percoll layer 4 (P4 neutrophil precursors) consisted of the most mature neutrophil precursors (high CD16 expression levels and a segmented nucleus) (19, 27).

First, Fc{alpha}RI and Fc{gamma}RI expression levels were determined (Fig. 1, A and B). In bone marrow, P2 neutrophil precursors expressed a high level of Fc{gamma}RI, which was down-regulated during differentiation. An ~3- ± 0.08-fold decrease in Fc{gamma}RI expression was observed in P4 neutrophil precursors compared with P2 precursors in each donor (n = 4). On mature blood neutrophils, the level of Fc{gamma}RI was low to absent (data not shown, n = 5), but could be up-regulated by G-CSF treatment. As such, experiments were only performed with G-CSF-stimulated blood neutrophils. Fc{alpha}RI expression levels were low on P2 neutrophil precursors and were up-regulated during differentiation. In each donor, a 2.7- ± 0.4-fold increase in Fc{alpha}RI expression was observed in P4 compared with P2 neutrophil precursors (n = 4). In blood, unstimulated as well as G-CSF neutrophils expressed high Fc{alpha}RI levels.


Figure 1
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  		FIGURE 1. Fc{alpha}RI and Fc{gamma}RI expression levels and neutrophil-mediated tumor cell killing. A, Surface expression of Fc{alpha}RI (left panel), or Fc{gamma}RI (right panel) in P2 (dotted line), P3 (thin line), and P4 (thick line) neutrophil precursors, as well as G-CSF neutrophils (dark filled area). Filled light gray area represents secondary Ab only. A representative example of eight is shown. B, Quantification of Fc{alpha}RI and Fc{gamma}RI expression on P2 ({square}), P3 (Figure 1), and P4 (Figure 1) neutrophil precursors, and on G-CSF neutrophils ({blacksquare}). Mean fluorescent intensities (MFI) ± SEM of four experiments are shown. C, Lysis of SK-BR-3 tumor cells by P2, P3, or P4 neutrophil precursors and G-CSF neutrophils in the presence of 2 µg/ml Fc{alpha}RI x Her-2/neu BsAb ({square}) or 2 µg/ml Fc{gamma}RI x Her-2/neu BsAb ({blacksquare}) measured in a 51Cr-release assay. One representative example of three experiments is shown. *, p < 0.05; **, p < 0.01; ns, nonsignificant.

 
The capacity of bone marrow neutrophils to lyse tumor cells via triggering of Fc{alpha}RI or Fc{gamma}RI was determined in 51Cr-release assays, in which SK-BR-3 tumor cells were used as targets. G-CSF-stimulated blood neutrophils were able to lyse tumor cells via triggering of Fc{gamma}RI, but lysis was more efficient by targeting of Fc{alpha}RI (Fig. 1C). P2 neutrophil precursors were unable to mediate tumor cell killing via either FcR. P3 neutrophil precursors, which coexpressed Fc{alpha}RI and Fc{gamma}RI, triggered tumor cell killing via Fc{alpha}RI, but not via Fc{gamma}RI. P4 neutrophil precursors, with low expression of Fc{gamma}RI, did not induce tumor cell lysis by targeting to Fc{gamma}RI either, whereas lysis was mediated after triggering Fc{alpha}RI.

Respiratory burst and FcR internalization on bone marrow neutrophils

We next investigated whether FcR-mediated tumor cell lysis was the sole effector function in which Fc{gamma}RI activation was absent, or whether other neutrophil functions were impaired as well. Therefore, FcR-mediated respiratory burst activity was analyzed. Cross-linking of Fc{alpha}RI or Fc{gamma}RI on G-CSF neutrophils induced respiratory burst activity, albeit the activity mediated via Fc{gamma}RI was consistently lower, compared with Fc{alpha}RI (Fig. 2A). Fc{alpha}RI triggering on P2, P3, and P4 neutrophil precursors activated a respiratory burst as well, although the maximal burst activities and durations were lower, compared with respiratory bursts observed in G-CSF neutrophils (1.5–4.0 x 106 cpm compared with 11.5 x 106 cpm, respectively; Fig. 2, B–D). In P2 neutrophil precursors, which expressed the highest Fc{gamma}RI level, cross-linking of Fc{gamma}RI induced some respiratory burst activity. This burst level, however, was minimal and reached only 0.5 x 106 cpm (Fig. 2B). Cross-linking of Fc{gamma}RI in P3 and P4 neutrophil precursors did not induce any respiratory burst activity (Fig. 2, C and D).


Figure 2
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  		FIGURE 2. Fc{alpha}RI- and Fc{gamma}RI-induced respiratory burst by neutrophils. Respiratory burst triggered after cross-linking Fc{alpha}RI (•), or Fc{gamma}RI ({square}) measured with a luminometer for 30 min in G-CSF neutrophils (A), and in P2 neutrophil precursors (B), P3 neutrophil precursors (C), or P4 neutrophil precursors (D). As a negative control, respiratory burst was measured in the presence of a control isotype Ab (+, dotted lines). A representative example of three separate experiments is shown.

 
Impaired FcR {gamma}-chain-mediated signaling by Fc{gamma}RI in neutrophil precursors

Furthermore, FcR {gamma}-chain-mediated signaling was investigated by triggering either receptor. As P2 neutrophil precursors express low Fc{alpha}RI levels, and P4 neutrophil precursors express low Fc{gamma}RI levels, we used P3 precursors, which coexpress both FcRs, for additional experiments. FcR cross-linking induces tyrosine phosphorylation of ITAM, which triggers activation of the PI3K pathway, leading to calcium mobilization (28). In G-CSF neutrophils, cross-linking of either Fc{alpha}RI or Fc{gamma}RI induced an increase in intracellular-free calcium levels (Fig. 3A). Although the total levels of Fc{alpha}RI and Fc{gamma}RI-mediated calcium release were similar (Fig. 3C), Fc{gamma}RI-mediated calcium mobilization was consistently slower compared with Fc{alpha}RI-mediated mobilization. On average, Fc{alpha}RI-mediated calcium mobilization peaked 29.8 ± 2.3 s after cross-linking, whereas Fc{gamma}RI-mediated calcium mobilization peaked after 48.5 ± 2.9 s (n = 4, p < 0.05, data not shown). In P3 neutrophil precursors, however, a rise in intracellular-free calcium was only observed after cross-linking Fc{alpha}RI (Fig. 3B). Fc{gamma}RI cross-linking did not result in release of intracellular calcium (Fig. 3C).


Figure 3
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  		FIGURE 3. Effect of FcR cross-linking on intracellular-free calcium levels. Intracellular-free calcium levels were measured after cross-linking Fc{alpha}RI (•) or Fc{gamma}RI ({square}) in G-CSF neutrophils (A) or P3 neutrophil precursors (B). Baseline calcium levels were established for 20 s, after which a cross-linking Ab was added (arrows). As a negative control, intracellular-free levels were measured in the presence of cross-linking Ab only (+, dotted lines). Calcium mobilization assays were repeated four times, yielding similar results. To quantify the levels of calcium mobilization, AUC of four separate experiments were determined and divided by the AUC of cross-linking Ab only, leading to a fold increase in calcium level compared with background (C). *, p < 0.05; ns, nonsignificant.

 
FcR cross-linking furthermore activates the RAS-ERK pathway, thereby triggering phosphorylation of MAPK (P-MAPK) (29). We previously showed that cross-linking of either Fc{alpha}RI or Fc{gamma}RI on G-CSF neutrophils induced MAPK phosphorylation, albeit P-MAPK levels were higher following triggering of Fc{alpha}RI, compared with Fc{gamma}RI (19). In P3 neutrophil precursors, Fc{alpha}RI cross-linking induced MAPK phosphorylation, which was detected within 15 min, and peaked after 60 min of cross-linking (Fig. 4). In contrast, cross-linking of Fc{gamma}RI on these precursors did not result in P-MAPK (Fig. 4).


Figure 4
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  		FIGURE 4. Effect of FcR cross-linking on MAPK phosphorylation. P3 neutrophil precursors were incubated with anti-Fc{alpha}RI, or anti-Fc{gamma}RI mAb, and cross-linked with a secondary Ab at 37°C for the indicated times (depicted in seconds). As a negative control, unlabeled neutrophils were incubated with anti-Fc{alpha}RI (A77), anti-Fc{gamma}RI (m22), or cross-linking Ab (indicated by "2nd") only. Western blots were stained for P-MAPK, stripped, and restained for total MAPK to assess protein loading. A representative example of three is shown, yielding similar results.

 
Efficient internalization of both Fc{alpha}RI and Fc{gamma}RI in neutrophil precursors

As all above-mentioned FcR {gamma}-chain-dependent functions were induced by Fc{alpha}RI but not by Fc{gamma}RI, we next performed an internalization assay, which is an FcR {gamma}-chain-independent function (24, 30). Cross-linking of either Fc{alpha}RI or Fc{gamma}RI on P3 neutrophil precursors induced efficient receptor internalization (Fig. 5, A and B). Within 5 min, both Fc{alpha}RI and Fc{gamma}RI levels decreased by 50–70% (Fig. 5C). These data therefore suggest that the discrepancy between Fc{alpha}RI and Fc{gamma}RI-mediated functions was restricted to the FcR {gamma}-chain pathway.


Figure 5
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  		FIGURE 5. FcR internalization in immature neutrophils. Fc{alpha}RI (A) or (B) Fc{gamma}RI surface expression levels before FcR cross-linking (filled area) and 20 min after FcR cross-linking (thick line) at 37°C. C, Receptor expression before cross-linking was set at 100%. Fc{alpha}RI (•) or Fc{gamma}RI ({square}) were cross-linked for the indicated time periods and remaining surface expression levels were determined. Percentage surface expression ± SEM of two experiments are shown.

 
Coimmunoprecipitation of FcR {gamma}-chain with Fc{alpha}RI or Fc{gamma}RI in neutrophil precursors

To evaluate association of both receptors with the FcR {gamma}-chain, we established a coimmunoprecipitation assay. When P3 and P4 neutrophil precursors were lysed using mild conditions with RIPA buffer, FcR {gamma}-chain was pulled down by Fc{alpha}RI (81.4 arbitrary units (A.U.) for P3, and 84.9 A.U. for P4 precursors) as well as via Fc{gamma}RI (68.9 for P3, and 76.4 A.U. for P4 precursors), indicating that both FcRs were associated with FcR {gamma}-chain (Fig. 6A). However, in a NP40 lysis buffer, FcR {gamma}-chain was coimmunoprecipitated via Fc{alpha}RI but not via Fc{gamma}RI (A.U. for P3 neutrophils are 94.1 and 2.3, respectively; A.U. for P4 neutrophils are 92.9 and 15.6, respectively), indicating that association of Fc{gamma}RI with FcR {gamma}-chain was abrogated using this detergent (Fig. 6B). In parallel experiments, similar results were observed with mature neutrophils (data not shown).


Figure 6
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  		FIGURE 6. Interaction of Fc{alpha}RI and Fc{gamma}RI with FcR {gamma}-chain in immature neutrophils. P3 and P4 neutrophil precursors were lysed with either RIPA buffer (A), or with a NP40 detergent (B), and immunoprecipitated with anti-Fc{alpha}RI, anti-Fc{gamma}RI, or irrelevant isotype control mAb (indicated by "IRR"). Samples were stained on Western blots for the FcR {gamma}-chain. Films were scanned with a densitometer and data were corrected for global background. One experiment of three is shown, yielding similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Triggering of neutrophil Fc{alpha}RI induces more efficient tumor cell killing, compared with Fc{gamma}RI (18, 19). Furthermore, immature neutrophils effectively lyse tumor cells via Fc{alpha}RI, but not Fc{gamma}RI (19). In the present study, we evaluated the mechanisms underlying the dissimilarities in FcR-mediated effector functions in different subsets of neutrophil precursors. We observed that ADCC, respiratory burst, and signaling functions like calcium mobilization and MAPK phosphorylation were effectively triggered by Fc{alpha}RI, but not by Fc{gamma}RI. FcR internalization, however, was readily induced by cross-linking either Fc{alpha}RI or Fc{gamma}RI. Formally, we cannot exclude the possibility that the observed differences are Ab-specific rather than receptor specific. However, this is less likely as the anti-Fc{alpha}RI and anti-Fc{gamma}RI Abs used in this study have similar affinities. Furthermore, Fc{alpha}RI and Fc{gamma}RI expression is similar on G-CSF stimulated as well as on P3 neutrophils. Targeting either Fc{alpha}RI or Fc{gamma}RI on G-CSF neutrophils induces signaling and function. However, when the same Abs are used to target P3 neutrophils, only Fc{alpha}RI mediates signaling. Targeting Fc{gamma}RI on P3 neutrophils with the same anti-Fc{gamma}RI Ab that induces signaling in G-CSF neutrophils does not result in any functional activity, supporting that Fc{gamma}RI signaling is hampered in immature neutrophils.

Interestingly, Fc{alpha}RI signaling is believed to be mediated through signaling routes that are also used by Fc{gamma}RI, requiring the common FcR {gamma}-chain (7, 8, 22, 23). Earlier work showed that most effector functions, such as phagocytosis and cytokine production, by either Fc{gamma}RI or Fc{alpha}RI were dependent on the ITAM-signaling motifs within this subunit (24, 31). However, a few FcR {gamma}-chain-independent functions have been described. Both Fc{alpha}RI and Fc{gamma}RI can mediate endocytosis without the FcR {gamma}-chain. This was shown for Fc{gamma}RI in transfection studies with COS cells (24, 32), and for Fc{alpha}RI in colostral neutrophils (33), which express a population of Fc{alpha}RI that is not associated with the FcR {gamma}-chain, but can mediate IgA endocytosis (30). Because FcR internalization was the only effector function that was not hampered via Fc{gamma}RI, our data suggest that the observed discrepancy between Fc{alpha}RI and Fc{gamma}RI-mediated function is the result of a difference between both FcRs in the FcR {gamma}-chain-signaling pathway. Furthermore, because early signaling events, like calcium mobilization and MAPK phosphorylation, were not initiated by Fc{gamma}RI, our data suggested that the interaction with the FcR {gamma}-chain was affected.

This hypothesis was supported by coimmunoprecipitation studies, in which we found a less stable interaction between Fc{gamma}RI and the FcR {gamma}-chain, compared with Fc{alpha}RI. This correlates well with earlier data in which Fc{alpha}RI was found to bear a positively charged amino acid residue on position 209, which associates with a negatively charged amino acid of the FcR {gamma}-chain, resulting in an electrostatic interaction within the transmembrane region (25). The positions of the positively charged amino acid residue was critical, as changing its position within the transmembrane region abrogated signaling and effector functions, except for FcR internalization, due to disturbance of the physical association between Fc{alpha}RI and FcR {gamma}-chain (34). Fc{gamma}RI lacks such a positively charged amino acid in its transmembrane region, which might underlie a weaker association with the FcR {gamma}-chain.

The FcR {gamma}-chain is required for stable Fc{gamma}RI expression, as expression was lost in the absence of the FcR {gamma}-chain (23). Furthermore, Fc{epsilon}RI and Fc{gamma}RIII were shown to compete for available FcR {gamma}-chain in mast cells (35), a phenomenon that was also suggested to occur in neutrophils for Fc{alpha}RI and Fc{gamma}RI, when both FcRs were maximally engaged (36). In addition, neutrophils express relatively low levels of FcR {gamma}-chain, compared with monocytes (37). Therefore, we postulate that due to a stronger association of Fc{alpha}RI with the FcR {gamma}-chain and limited availability of FcR {gamma}-chain in immature neutrophils, Fc{alpha}RI competes with Fc{gamma}RI for available FcR {gamma}-chain. This consequently leads to inability of immature neutrophils to signal via Fc{gamma}RI, as well as to loss of Fc{gamma}RI expression during maturation. As Fc{gamma}RI triggering in P2 neutrophil precursors induced a small respiratory burst, some FcR {gamma}-chain might have been available for Fc{gamma}RI due to low Fc{alpha}RI expression levels in these early precursors.

In G-CSF neutrophils, FcR {gamma}-chain protein levels, measured by Western blotting, were considerably increased compared with levels in bone marrow and unstimulated blood neutrophils (data not shown, n = 3), indicating that more FcR {gamma}-chain is available after G-CSF stimulation. This might lead to the observed up-regulation of Fc{gamma}RI expression and function in G-CSF neutrophils (Fig. 1 and Ref. 20). We speculate that loss of Fc{gamma}RI expression on mature neutrophils is favorable for maintaining homeostasis, as Fc{gamma}RI is a high-affinity receptor for IgG (1) and, as such, may activate unwanted inflammatory responses. However, during bacterial infections, Fc{gamma}RI-expressing immature neutrophils are recruited from the bone marrow, which may help to clear the infection (38). Neutrophils of patients with streptococcal pharyngitis were shown to express increased numbers of Fc{gamma}RI (38).

Studies with Fc{alpha}RI x Fc{gamma}RI double transgenic mice supported the above-mentioned observations because, similar to human neutrophil precursors, mouse bone marrow neutrophils were unable to mediate ADCC via triggering of Fc{gamma}RI, but not Fc{alpha}RI (19). However, bone marrow neutrophils from G-CSF-treated mice could mediate ADCC via triggering of Fc{gamma}RI, as well (data not shown, n = 4).

Taken together, our data suggest that inefficient signaling and effector functions via Fc{gamma}RI on neutrophils is most likely induced by differences in interaction between Fc{alpha}RI or Fc{gamma}RI with the common FcR {gamma}-chain. This may lead to competition between both FcRs for available FcR {gamma}-chain in favor of Fc{alpha}RI, hereby abrogating Fc{gamma}RI function.


   Acknowledgments
 
We thank Eefke Petersen and Faiz Ramjankhan for provision of blood and bone marrow samples, Martin Glennie and Allison Tutt for generation of Fc{alpha}RI x Her-2/neu BsAb, and Paul Coffer, Erik-Jan Oudijk, Jantine Bakema, Jeffrey Beekman, and Olivier van Beekum for helpful discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by the Dutch Cancer Society (UU2001-2431) and the Netherlands Organization for Scientific Research (VENI 916.36.079). Back

2 Address correspondence and reprint requests to Dr. Marjolein van Egmond, Department of Molecular Cell Biology and Immunology, Vrije University Medical Center, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. E-mail address: M.vanEgmond{at}vumc.nl Back

3 Abbreviations used in this paper: ADCC, Ab-dependent cellular cytotoxicity; m, murine; BsAb, bispecific Ab; PO, peroxidase; P-MAPK, phosphorylated MAPK; A.U., arbitrary unit; AUC, area under the curve; NP40, Nonidet P-40; RIPA, radioimmunoprecipitation. Back

Received for publication December 18, 2006. Accepted for publication June 22, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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