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The Journal of Immunology, 1998, 161: 3666-3674.
Copyright © 1998 by The American Association of Immunologists

Modulation of Human Neutrophil Apoptosis by Immune Complexes1

Romina Gamberale2,*,{dagger}, Mirta Giordano*,{dagger}, Analía S. Trevani*,{dagger}, Graciela Andonegui* and Jorge R. Geffner*,{dagger}

* Laboratory of Immunology, Institute of Hematologic Research, National Academy of Medicine, and {dagger} Department of Microbiology, Buenos Aires University School of Medicine, Buenos Aires, Argentina


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study we examined whether immune complexes (IC) are able to modulate human neutrophil apoptosis. We observed different effects depending on the type of IC employed. Precipitating IC (pIC) and Ab-coated erythrocytes (E-IgG) triggered a marked stimulation of apoptosis, while heat-aggregated IgG and soluble IC, significantly delayed spontaneous apoptosis. Blocking Abs directed to Fc{gamma} receptor type II (Fc{gamma}RII), but not to Fc{gamma}RIII, markedly diminished the acceleration of apoptosis triggered by either pIC or E-IgG, supporting a critical role for Fc{gamma}RII in apoptosis stimulation. This phenomenon, on the other hand, does not appear to involve IC phagocytosis or the participation of CR3. Acceleration of neutrophil apoptosis triggered by either pIC or E-IgG seems to require the activation of the respiratory burst, as suggested by 1) the ability of catalase to prevent apoptosis stimulation; 2) the effect of azide, an heme enzyme inhibitor, which dramatically enhanced apoptosis induced by pIC or E-IgG; and 3) the inability of pIC or E-IgG to accelerate apoptosis of neutrophils isolated from CGD patients. It is well established that IC affect the course of inflammation by inducing the release of inflammatory cytokines, proteolytic enzymes, oxidative agents, and other toxic molecules. Our results suggest that IC may also affect the course of inflammation by virtue of their ability to modulate neutrophil apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Granulocyte neutrophils are short-lived cells, with a half-life of <24 h. In the absence of appropriate stimuli, neutrophils undergo characteristic changes indicative of programmed cell death or apoptosis, including cell shrinkage, nuclear chromatin condensation, and DNA fragmentation into nucleosome-length fragments (1, 2). As a first line of defense against host insult, neutrophils are rapidly recruited to inflammatory sites, where the expression of their apoptotic program can be modified by a number of agents. Granulocyte-macrophage CSF, IL-1, IL-2, and LPS have been shown to inhibit neutrophils apoptosis and prolong their functional life span (3, 4, 5, 6). Disparate results, on the other hand, have been reported regarding the effects of C5a, FMLP, granulocyte CSF, and IL-6 (3, 4, 5, 6, 7, 8). Apoptosis, which represents an alternative fate to necrosis, not only determines neutrophil uptake by macrophages, but is also associated with a loss of neutrophil functions, such as chemotaxis, phagocytosis, degranulation, and respiratory burst (7, 8, 9). For these reasons, neutrophil apoptosis may be considered a mechanism that contributes to the resolution of inflammation (1, 2, 7, 9).

Receptors for the constant region of IgG (Fc{gamma}R)3 fall into three major classes: Fc{gamma}RI (CD64), Fc{gamma}RII (CD32), and Fc{gamma}RIII (CD16). They comprise nine membrane-associated and three soluble Fc{gamma}R molecules, produced by alternative splicing of Fc{gamma}R transcripts or by proteolysis of surface receptors. Most membrane-associated Fc{gamma}R exist as hetero-oligomeric complexes with a ligand-binding {alpha}-chain, which determines affinity and isotype specificity, and a signaling component comprising {gamma}-, ß-, or {xi}-chains (10, 11, 12, 13).

Human neutrophils constitutively express two low affinity Fc{gamma}R: Fc{gamma}RIIa (10,000–20,000 sites/cell) and Fc{gamma}RIIIb (100,000–200,000 sites/cell). Fc{gamma}RIIa is a transmembrane molecule, while Fc{gamma}RIIIb is linked via a glycosylphosphatidyl inositol anchor to the cell membrane (10, 11, 12, 13, 14, 15). A large number of studies has examined the ability of neutrophil Fc{gamma}R to trigger inflammatory responses as a consequence of their interaction with immune complexes (IC). Phagocytosis, Ab-dependent cellular cytotoxicity, and secretion of soluble inflammatory mediators such as free radical oxidants, lysosomal enzymes, and cytokines can be triggered through Fc{gamma}R aggregation (16, 17, 18, 19). To our knowledge, no previous work has examined the effect of Fc{gamma}R engagement on neutrophil survival.

In the present study we analyze whether the activation of Fc{gamma}R by IC is able to modulate human neutrophil apoptosis. Our results show that different types of IC exert opposite effects. Thus, precipitating immune complexes (pIC) and Ab-coated erythrocytes (E-IgG) induce a dramatic stimulation of neutrophil apoptosis, while heat-aggregated IgG (aIgG) and soluble IC (sIC) significantly delay apoptotic cell death.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

The following drugs were used: acridine orange, ethidium bromide, propidium iodide, cytochalasin B, luminol, catalase (from bovine liver, 50,000 U/mg protein), and superoxide dismutase (from bovine erythrocytes, 5,000 U/mg protein; Sigma, St. Louis, MO). mAb 3G8 (IgG1), which recognizes human Fc{gamma}RIII, and IV.3 (IgG2b), which recognizes human Fc{gamma}RII, were obtained from Medarex (West Lebanon, NH). mAb IgM anti-Fas (CH-11) and IgG1 anti-FasL (G247-4 and NOK-1) were obtained from PharMingen (San Diego, CA). Blocking Abs directed to CD11b (Mo1) and CD18 (IB4) were obtained from Immunotech (Marseille, France). For blocking studies, neutrophils were preincubated with the corresponding mAb during 30 min at 4°C. Concentrations of mAb three- to fivefold higher than those needed to saturate all binding sites (1–10 µg/ml), as determined by FACS analysis, were used in these studies.

Preparation of immune complexes

The pIC and sIC were prepared as we previously described (20), using human IgG or OVA (Sigma) as Ag and specific rabbit IgG Abs. The pIC were formed at the equivalence zone, and the sIC were formed at fivefold Ag excess, based on equivalence points determined by quantitative precipitin curves. In all cases, Ag and Ab were incubated for 1 h at 37°C and 18 h at 4°C. After this period, IC were centrifuged at 3,000 x g for 10 min, and the precipitate or the supernatant was recovered. The IgG aggregates were prepared by heating human IgG at a concentration of 5 mg/ml for 12 min at 63°C. Then, heat-aggregated human IgG was centrifuged at 10,000 x g for 5 min, and the precipitate was discarded. IgG-coated erythrocytes were prepared using mouse erythrocytes (MRBC; 1% (v/v) in RPMI 1640 with 1% heat-inactivated FCS (Difco, Detroit, MI) sensitized with subagglutinating amounts of rabbit IgG anti-MRBC.

Blood samples

Blood samples were obtained from healthy donors who had taken no medication for at least 10 days before the day of sampling. Blood was obtained by venipuncture of the forearm vein and was drawn directly into heparinized plastic tubes.

Neutrophil isolation

Neutrophils were isolated by Ficoll-Hypaque gradient centrifugation (Ficoll Pharmacia, Uppsala, Sweden; Hypake, Winthrop Products, Buenos Aires, Argentina) and dextran sedimentation, as previously described (21). Contaminating erythrocytes were removed by hypotonic lysis. After washing, the cells (>96% neutrophils on May Grunwald/Giemsa-stained Cytopreps) were resuspended in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 1% heat-inactivated FCS. In another set of experiments, neutrophils were purified through two alternative procedures. One of them involved neutrophil isolation from heparinized blood samples by dextran sedimentation followed by centrifugation for 5 min at 50 to 70 x g. These cell suspensions contained mononuclear cells (15–35%) and neutrophils (65–85%). Contaminating erythrocytes were not removed; therefore, they were usually present at concentrations 5- to 15-fold higher than those of leukocytes. The second isolation procedure involved erythrocyte sedimentation by adding 40% (v/v) autologous plasma to the blood. The leukocyte-rich plasma was layered onto a discontinuous Percoll gradient, as previously described (22), and centrifuged at 600 x g for 20 min. The neutrophil-containing band was collected and washed with PBS. Cells were then resuspended in RPMI 1640 supplemented with 1% heat-inactivated FCS.

Quantitation of cellular apoptosis and viability by fluorescence microscopy

Quantitation was performed as previously described (23, 24), using the fluorescent DNA-binding dyes acridine orange (100 µg/ml) to determine the percentage of cells that had undergone apoptosis and ethidium bromide (100 µg/ml) to differentiate between viable and nonviable cells. With this method, nonapoptotic cell nuclei show variations in fluorescent intensity that reflect the distribution of euchromatin and heterochromatin. By contrast, apoptotic nuclei exhibit highly condensed chromatin that is uniformly stained by acridine orange. To assess the percentage of cells showing morphologic features of apoptosis, at least 200 cells were scored in each experiment.

Quantitation of neutrophil apoptosis by propidium iodide staining and flow cytometry

The proportion of neutrophils that displayed a hypodiploid DNA peak, i.e., apoptotic cells, was determined using a modification of Nicoletti’s protocol (25). Briefly, cell pellets containing 2.5 x 106 neutrophils were suspended in 400 µl of hypotonic fluorochrome solution (50 µg/ml propidium iodide in 0.1% sodium citrate plus 0.1% Triton X-100) and incubated for 2 h at 4°C. The red fluorescence of propidium iodide of individual nuclei was measured using a FACScan flow cytometer (Becton Dickinson Immunocytometry System, San Jose, CA). The forward scatter and side scatter of particles were simultaneously measured. Cell debris was excluded from analysis by appropriately raising the forward scatter threshold. The red fluorescence peak of neutrophils with normal (diploid) DNA content was set at channel 250 in the logarithmic mode. Apoptotic cell nuclei emitted fluorescence in channels 4 through 200.

Measurement of fluctuations in intracellular Ca2+ concentrations ([Ca2+]i)

Changes in intracellular calcium concentrations [Ca2+]i were monitored using fluo-3/AM, as previously described (26). Briefly, neutrophils, suspended at a concentration of 5 x 106 cells/ml in RPMI 1640 were incubated with 4 µM fluo-3/AM for 30 min at 30°C. Then, loaded cells were washed twice and resuspended at 5 x 106 cells/ml in RPMI 1640 supplemented with 5% heat-inactivated FCS. Aliquots of 50 µl of this cell suspension were then added to 450 µl of RPMI 1640 medium containing 5% FCS and 1 mM CaCl2 and warmed at 37°C. The samples were immediately loaded onto the flow cytometer, and the basal fluorescence (FL1) was recorded during 30 s. Then, cells were stimulated with IC, and the fluorescence was recorded during an additional 400 s. Acquisition of samples was performed at 37°C. Fluctuations in cytoplasmic free calcium concentrations were recognized as alterations in fluo-3 fluorescence intensity over time. Data were analyzed by employing CellQuest software (Becton Dickinson, Mountain View, CA). A gate based on forward and side scatters was used to exclude debris. Cells that raised their [Ca2+]i, in response to IC, to higher levels than that shown by 97% of resting cells were considered to be activated.

Chemiluminescence assays

Luminescence responses of neutrophils (2.5 x 106/ml in culture medium supplemented with 1% heat-inactivated FCS) triggered by IC were measured with a whole blood Lumi aggregometer (Chrono-Log) at 1000 rpm and 37°C in the presence of luminol (10-7 M), as we previously described (27). In all cases, light emission was continuously registered for 10 min. Data are expressed as the maximum response observed during this period in relative chemiluminescence units (URCL). One CL unit was defined as one centimeter shifting of the light emission signal on the paper recorder.

Phagocytosis assay

The phagocytosis assay was performed as previously described (28). Briefly, neutrophils were suspended at a concentration of 2.5 x 106/ml in culture medium supplemented with 1% heat-inactivated FCS. One hundred microliters of this suspension was mixed with 50 µl of 51Cr-labeled mouse erythrocytes (5 x 108/ml) sensitized with subagglutinating concentrations of rabbit IgG anti-MRBC. The optimal concentration of rabbit IgG anti-MRBC was determined in preliminary experiments and was the highest antiserum dilution (1/512) that yielded maximal phagocytic activity by neutrophils. After incubation for 30 min at 37°C in 5% CO2-95% humidified air, the noningested MRBC were lysed with 0.83% NH4Cl. Ingested MRBC were detected in a gamma counter. Phagocytosis was recorded as the phagocytic index (number of MRBC ingested by 100 phagocytes). No phagocytosis was detected when neutrophils were incubated with unsensitized MRBC.

Statistical analysis

Student’s paired t test was used to determine the significance of differences between means, and p < 0.05 was taken as indicating statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immune complexes modulate neutrophil apoptosis

Immune complexes constitute a heterogeneous group of Fc{gamma}R ligands that differ not only in their ability to trigger distinct cellular responses but also in the mechanisms by which they induce cell activation (10, 16, 17, 22, 29, 30). These observations prompted us to use different types of IC to analyze the impact of Fc{gamma}R ligands on neutrophil apoptosis. The IC employed were sIC prepared with OA and rabbit IgG Abs anti-OA (sIC), human heat-aggregated IgG (aIgG), pIC prepared with human IgG as Ag and rabbit IgG Abs to human IgG, and mouse erythrocytes coated with specific rabbit IgG Abs (E-IgG). In a first set of experiments, apoptosis was revealed after 18 h of culture at 37°C by fluorescence microscopy, using the fluorescent DNA-binding dye acridine orange. As shown in Figure 1Go, treatment with either sIC or aIgG induced a significant delay of apoptosis. By contrast, treatment with pIC or E-IgG increased the apoptotic rate of neutrophils (Fig. 1Go). Similar results were obtained when pIC and E-IgG were prepared using OA and rabbit IgG Abs anti-OA, and sheep erythrocytes and specific rabbit IgG Abs, respectively (data not shown). Control cells and cells treated with either sIC or aIgG showed percentages of viability always >94%, as judged by the exclusion of both trypan blue and the fluorescent dye ethidium bromide. In contrast, neutrophils treated with pIC or E-IgG displayed significant levels of necrosis (range, 11–39%; n = 13–17).



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  		FIGURE 1. Modulation of neutrophil apoptosis by IC. Neutrophils (2.5 x 106/ml) were cultured for 18 h at 37°C in the presence of pIC (10 µg/ml), E-IgG (1%, v/v), aIgG (50 µg/ml), or sIC (50 µg/ml). Then, the percentage of apoptotic cells was determined by fluorescence microscopy. Results are expressed as the mean ± SEM of nine experiments. *, Statistical significance (p < 0.01) compared with the control.

 
To explore the time course of neutrophil apoptosis induced by pIC and E-IgG, it was evaluated after 8 and 12 h of culture. At these times, both stimuli induced high levels of apoptosis without affecting cell viability, which was always >94% (Fig. 2Go). On the other hand, when apoptosis was assessed after 3 h of culture, <10% of the neutrophil population were apoptotic, and no significant differences were observed between untreated and IC-treated cells. Regarding the effects of sIC and aIgG, there were no significant differences in the percentages of apoptosis between untreated and treated neutrophils at either 8 or 12 h of culture (data not shown). Unless otherwise stated, subsequent experiments to analyze the ability of pIC and/or E-IgG to stimulate neutrophil apoptosis were performed after incubation for 12 h.



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  		FIGURE 2. Stimulation of neutrophil apoptosis by pIC or E-IgG. Neutrophils (2.5 x 106/ml) were cultured for 8 h (open bars) or for 12 h (hatched bars) at 37°C in the presence of pIC (10 µg/ml) or E-IgG (1%, v/v). Then, the percentage of apoptotic cells was determined by flow cytometry (A) and fluorescence microscopy (B). A, Histograms of a representative experiment (n = 7), revealed at 12 h of incubation, showing the percentage (M1) of nuclei with hypodiploid DNA content. B, Results are expressed as the mean ± SEM of seven experiments. *, Statistical significance (p < 0.01) compared with controls.

 
It is noteworthy that pIC and E-IgG exerted stimulatory effects on neutrophil apoptosis at very low concentrations. Thus, 1 µg/ml of pIC significantly increased the apoptotic rate of neutrophils (apoptosis after 12 h of culture, 9 ± 4 vs 28 ± 7%, untreated vs pIC-treated cells, mean ± SEM; n = 5; p < 0.05). Similarly, E-IgG used at 0.2% (v/v) were also effective (apoptosis after 12 h of culture, 7 ± 4 vs 21 ± 5%, untreated vs E-IgG-treated cells, mean ± SEM; n = 5, p < 0.05).

Previous observations suggest that standard preparative techniques using a Ficoll-Hypaque gradient and lysis of contaminating erythrocytes may result in neutrophil activation, loss of membrane integrity, and/or loss of cells during aging by clumping (31, 32). To rule out the possibility that our results could be related to the induction of cell injury or "priming" during purification, we performed experiments with neutrophils obtained by two alternative isolation procedures, as described in Materials and Methods. Cellular suspensions obtained through dextran sedimentation of blood samples contained mononuclear cells (15–35%), neutrophils (60–85%), and erythrocytes. Treatment of these cells with either pIC or E-IgG induced apoptotic rates similar to those observed in neutrophils purified by standard preparative techniques (neutrophil apoptosis evaluated by fluorescence microscopy after 12 h of culture, 6 ± 3, 59 ± 7, and 48 ± 6% for untreated, pIC-treated, and E-IgG-treated neutrophils, respectively, mean ± SEM; n = 5; p < 0.01, untreated vs treated cells). In contrast to what was found in neutrophils, apoptosis of mononuclear cells was not modify by IC treatment; it was always <4%. Finally, experiments were performed using neutrophils isolated from leukocyte-rich plasma by discontinuous Percoll gradient. The results obtained were comparable to those described above (neutrophil apoptosis revealed by fluorescence microscopy after 12 h of culture, 8 ± 3, 68 ± 6, and 52 ± 5% for untreated, pIC-treated, and E-IgG-treated neutrophils, respectively, mean ± SEM; n = 5; p < 0.01, untreated vs treated cells).

Stimulation of apoptosis by pIC and E-IgG is mediated through Fc{gamma}RIIa and does not require IC phagocytosis

The roles of Fc{gamma}RIIa and Fc{gamma}RIIIb in the stimulation of apoptosis by pIC and E-IgG were evaluated using blocking mAb directed to Fc{gamma}RIIa (IV.3) and Fc{gamma}RIIIb (3G8). Incubation of neutrophils with these mAb had no effect on spontaneous apoptosis. As shown in Figure 3Go, the acceleration of apoptosis triggered by either pIC or E-IgG was markedly diminished by mAb IV.3, while it was not modified by mAb 3G8.



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  		FIGURE 3. Role of Fc{gamma}R in the stimulation of apoptosis by pIC or E-IgG. Neutrophils (2.5 x 106/ml) were cultured 30 min at 4°C alone (open bars) or in the presence of IV.3 (anti-Fc{gamma}RII; hatched bars) or 3G8 (anti-Fc{gamma}RIII; stippled bars). Then, pIC (10 µg/ml) or E-IgG (1%, v/v) were added, and apoptosis was revealed by fluorescence microscopy after 12 h of incubation at 37°C. Results are expressed as the mean ± SEM of five experiments. *, Statistical significance (p < 0.01) compared with neutrophils cultured without anti-Fc{gamma}R Abs.

 
Recent works have shown that neutrophils undergo apoptosis following phagocytosis of bacteria (33, 34). To examine whether phagocytosis of pIC or E-IgG was responsible for the stimulation of apoptosis, we performed experiments in the presence of cytochalasin B, a microfilament-disrupting agent. It was found that cytochalasin B, at concentrations that almost completely abrogate phagocytosis of E-IgG (5 µg/ml; inhibition of phagocytosis, 89 ± 6%, mean ± SEM; n = 4), had no effect on the stimulation of neutrophil apoptosis induced by either pIC or E-IgG (apoptosis after 12 h of culture, 14 ± 4, 78 ± 7, and 69 ± 5% for untreated, pIC-treated, and E-IgG-treated neutrophils, respectively, mean ± SEM; n = 5).

Promotion of apoptosis induced by pIC and E-IgG does not involve the Fas/FasL system

The Fas/FasL system has been implicated as an important cellular pathway mediating apoptosis in different cell types (35). Neutrophils constitutively express not only Fas, but also FasL, which appears to be responsible at least in part for their rapid rate of spontaneous apoptosis (36).

Previous observations in NK cells showed that Fc{gamma}R stimulation of activated cells results in the transcriptional up-regulation of FasL, a mechanism that facilitates subsequent autocrine NK cell apoptosis (37). In addition, Andrés and co-workers (38) have demonstrated that stimulation of Fc{gamma}R induces apoptosis of murine eosinophils, a phenomenon associated with the induction of Fas expression. Taking these data into account, we examined whether pIC were able to increase the expression of Fas and/or FasL in neutrophils by using the mAb CH-11 directed to Fas, and the mAb G247-4 directed to FasL. After 3 h of incubation with pIC, there was no difference in the expression of Fas or FasL between untreated and pIC-treated neutrophils (Fig. 4Go). Similar results were observed in E-IgG-treated neutrophils (data not shown). These results suggest that the Fas/FasL system is not involved in the acceleration of apoptosis triggered by either pIC or E-IgG. This conclusion was further supported by the fact that treatment of neutrophils with a blocking mAb directed to FasL (NOK-1) did not impair the acceleration of apoptosis induced by either pIC or E-IgG (apoptosis after 12 h of culture, 7 ± 3, 67 ± 9, and 48 ± 7% for untreated, pIC-treated, and E-IgG-treated neutrophils, respectively, mean ± SEM; n = 3).



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  		FIGURE 4. Expression of Fas and FasL in neutrophils stimulated by pIC. Neutrophils (2.5 x 106/ml) were treated with pIC (10 µg/ml) for 3 h at 37°C. Then, the expression of Fas (a) and FasL (b) was evaluated by flow cytometry. Histograms of a representative experiment (n = 4) are depicted.

 
Promotion of apoptosis induced by pIC and E-IgG does not involve CR3

Recently, Walzog et al. (22) have shown that cross-linking of CR3 (CD11b/CD18, Mac-1, {alpha}mß2) promotes apoptosis in neutrophils that have been activated by either treatment with TNF-{alpha} or migration through an endothelial cell monolayer. Coxon et al. (39), on the other hand, found that apoptosis of extravasated peritoneal neutrophils in vitro is delayed in mice selectively deficient in CR3, and that CR3-mediated phagocytosis and the associated oxidative burst lead to rapid neutrophil apoptosis. Taking this into account and considering that neutrophil CR3 cooperates with Fc{gamma}R in the induction of different responses triggered by IgG IC such as adhesion, phagocytosis, and respiratory burst (40, 41, 42, 43), we analyzed whether acceleration of apoptosis induced by pIC or E-IgG involved a CR3-dependent pathway. Experiments were performed using neutrophils treated with blocking mAb directed to either CD11b (Mo1) or CD18 (IB4) (44, 45). The results presented in Figure 5Go show that these mAb did not prevent the acceleration of apoptosis induced by pIC or E-IgG.



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  		FIGURE 5. Role of CR3 in the stimulation of apoptosis by pIC or E-IgG. Neutrophils (2.5 x 106/ml) were cultured for 30 min at 4°C alone (open bars) or in the presence of Mo1 (anti-CD11b; hatched bars) or IB4 (anti-CD18; stippled bars). Then, pIC (10 µg/ml) or E-IgG (1%, v/v) were added, and apoptosis was revealed by fluorescence microscopy after 12 h of incubation at 37°C. Results are expressed as the mean ± SEM of five experiments.

 
Comparison between the abilities of aIgG and pIC to trigger transient elevations of cytosolic Ca2+ and to activate the neutrophil respiratory burst

Elevation of cytosolic Ca2+ stimulates apoptosis in different cell types (46). However, in the neutrophil, transient elevation of Ca2+ exerts an inhibitory effect on apoptosis (47). Taking these data into account, we next examined whether the opposite effects of aIgG and pIC on neutrophil apoptosis could be related at least in part to differences in their ability to trigger a rise in intracellular Ca2+ concentrations. As shown in Figure 6Go, both stimuli induced similar fluctuations in intracellular Ca2+ concentrations.



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  		FIGURE 6. Fluctuations in intracellular Ca2+ concentrations induced by pIC or aIgG. Neutrophils were loaded with fluo-3/AM as described in Materials and Methods. The basal level of fluo-3 fluorescence was recorded for about 30 s. The pIC (a) or aIgG (b) were added as indicated, and fluorescence was recorded for 400 s. The increase in [Ca2+]i was recognized as an increase in fluo-3 fluorescence. Results from a representative experiment (n = 5) are depicted.

 
Increasing evidence suggests that oxidative stress plays an important role in the induction of apoptosis in different cell types, including neutrophils (33, 39, 48, 49). Many of the agents able to stimulate apoptosis are either oxidants or stimulators of cellular oxidative metabolism. Conversely, many inhibitors of apoptosis have antioxidant activities or increase cellular antioxidant defenses (48, 49, 50, 51, 52). Taking these observations into account, we next compared the abilities of pIC and aIgG to trigger the neutrophil respiratory burst, measured as CL emission. The results presented in Figure 7Go show that CL responses triggered by pIC were around 10-fold higher than those induced by aIgG. More importantly, when the time course of CL was studied, it was found that light emission induced by aIgG declined to basal values at early as 3 min after stimulation. By contrast, light emission triggered by pIC remained unchanged for at least 2 h after stimulation (CL, 94 ± 6 vs 89 ± 8 URCL; 5 vs 120 min, mean ± SEM; n = 5). In a separate set of experiments we examined whether CL responses induced by sIC and E-IgG were comparable to those induced by aIgG and pIC, respectively. It was found that sIC triggered low responses (6 ± 2 URCL, mean ± SEM; n = 5) that declined to basal levels 4 min after stimulation, while E-IgG (1%, v/v) triggered high responses (47 ± 5 URCL, mean ± SEM; n = 5) that decreased to basal levels 30 min after stimulation. In all cases, CL responses were markedly inhibited (inhibition, >85%) when conducted in the presence of catalase (1000 U/ml) and superoxide dismutase (SOD; 1000 U/ml), indicating that light emission was ascribed to the generation of oxygen-reactive intermediates (IRO).



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  		FIGURE 7. Chemiluminescence responses of neutrophils stimulated by pIC or aIgG. Neutrophils (2.5 x 106/ml) were triggered by pIC (10 µg/ml) or aIgG (50 µg/ml), and light emission was continuously recorded for 10 min. Data are expressed as the maximum response observed during this period, in relative chemiluminescence units (URCL). Values are the mean ± SEM of six experiments.

 
Effect of IRO scavengers on the stimulation of neutrophil apoptosis induced by pIC

A role for IRO in the acceleration of apoptosis triggered by pIC was examined using catalase and SOD. The results presented in Figure 8Go show that catalase, but not SOD, prevented the stimulation of apoptosis triggered by pIC, suggesting a critical role for hydrogen peroxide. Comparable results were obtained using E-IgG as a triggering stimuli for apoptosis (apoptosis after 12 h of culture, 64 ± 7 and 31 ± 5%, mean ± SEM; n = 4; for neutrophils cultured for 12 h in the absence or the presence of 5000 U/ml catalase).



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  		FIGURE 8. Effects of catalase and SOD on the stimulation of neutrophil apoptosis induced by pIC. Untreated and pIC-treated neutrophils (2.5 x 106/ml) were incubated in the presence of catalase (5000 U/ml; hatched bars) or SOD (5000 U/ml; stippled bars) or with medium alone (open bars) for 12 h at 37°C. Then, the percentage of apoptotic cells was determined by fluorescence microscopy. Results are expressed as the mean ± SEM of six experiments. *, Statistical significance (p < 0.01) compared with pIC-treated neutrophils cultured in the absence of catalase.

 
Previous reports have indicated that when neutrophil catalase and MPO activities were inhibited by heme enzyme inhibitors there was a significant increase in both O2 consumption and H2O2 release during neutrophil activation (53, 54). To further explore the role of H2O2 in the acceleration of neutrophil apoptosis induced by either pIC or E-IgG, we analyzed the effect of sodium azide (0.1 mM), a heme enzyme inhibitor. Sodium azide did not affect the spontaneous apoptotic rate of neutrophils revealed at either 12 or 18 h of culture (apoptosis, 10 ± 4 vs 12 ± 5%, and 34 ± 6 vs 37 ± 5%, respectively, control vs sodium azide, mean ± SEM; n = 5). In contrast, when neutrophils were stimulated with either pIC or E-IgG, azide induced a dramatic increase in the apoptotic rates (apoptosis revealed at 3 h of culture, 5 ± 4 vs 82 ± 7% for neutrophils treated with pIC in the absence or the presence of azide, respectively, and 5 ± 3 vs 49 ± 6% for neutrophils treated with E-IgG in the absence or the presence of azide, respectively, mean ± SEM; n = 4). It should be emphasized that quantitation of apoptosis was performed at 3 h of culture, since after this time, neutrophils stimulated by either pIC or E-IgG in the presence of azide showed high levels of necrosis (data not shown).

Effects of aIgG, pIC, and E-IgG on apoptotic rates of neutrophils from patients with chronic granulomatous disease (CGD)

To further analyze whether the production of IRO was responsible for the stimulation of neutrophil apoptosis by pIC and E-IgG, assays were performed using neutrophils isolated from three patients with CGD, a rare hereditary disorder characterized by a diminished or absent production of IRO due to a defect in any one of the components of NADPH oxidase (55). Preliminary studies showed that neutrophils isolated from these patients produced, in response to FMLP, pIC, and zymosan, O2- levels <5% those produced by normal cells (data not shown).

When the effects of pIC and E-IgG on neutrophil apoptosis were examined, it was found that neither pIC nor E-IgG induced stimulation of apoptosis in CGD neutrophils after 12 or 18 h of incubation (Fig. 9Go). Indeed, a nonstatistically significant delay of apoptosis was observed with E-IgG, which was similar to that induced by aIgG. The fact that pIC and E-IgG were unable to accelerate apoptosis of CGD neutrophils could not be attributable to an impairment in their capacity to bind to Fc{gamma}R. Thus, rosettes formation with E-IgG, binding of aIgG, as well as induction of transient elevations in [Ca2+]i triggered by pIC were comparable in both CGD and normal neutrophils (data not shown).



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  		FIGURE 9. Effect of aIgG, pIC, or E-IgG on apoptosis of neutrophils from CGD patients. Neutrophils (2.5 x 106/ml) were cultured for 12 or 18 h at 37°C in the presence of aIgG (50 µg/ml; hatched bars), pIC (10 µg/ml; stippled bars), or E-IgG (1%, v/v; black bars) or with medium alone (open bars). Then, the percentage of apoptotic cell was determined by fluorescence microscopy. Results are expressed as the mean ± SEM of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The experiments presented here demonstrate that neutrophil apoptosis can be modulated by IC. Stimulatory or inhibitory effects were observed depending on the characteristics of the IC employed. Thus, while pIC and E-IgG induced a dramatic stimulation of apoptosis, sIC or aIgG, in contrast, significantly delayed spontaneous neutrophil apoptosis. These results suggest that neutrophil Fc{gamma}R can trigger transductional pathways involved not only in inflammatory, cytotoxic, and hypersensitivity functions, but also in the control of cell survival.

A growing body of evidence shows that the engagement of neutrophil Fc{gamma}R results in the activation of different responses depending on the composition of the IC (10, 16, 29, 30). Our data showing opposite effects induced by different Fc{gamma}R ligands on neutrophil apoptosis are consistent with these observations. Moreover, we found differences between particulate (pIC and E-IgG) and soluble (sIC and aIgG) Fc{gamma}R ligands with regard to other neutrophil functions. Thus, particulate and soluble Fc{gamma}R ligands were equally effective to trigger both an increase in intracellular Ca2+ concentrations (Fig. 5Go) and a neutrophil shape change (data not shown); however, they dramatically differed in their capacities to activate the respiratory burst. We also observed differences in their abilities to trigger the production of cytokines; pIC, but not sIC or aIgG, was able to stimulate TNF-{alpha} synthesis (not shown). A possible explanation for these contrasting effects is that different signal intensities might be required to induce distinct Fc{gamma}R-dependent responses. A similar hypothesis was previously postulated to explain the behavior of the chemotactic peptide FMLP receptors. It was found that increasing levels of receptor occupancy generate neutrophil responses in a definitive order: cell polarization and actin polymerization (chemotaxis) before Ca2+ flux, before degranulation (56). On the other hand, experiments with small peptides, used as C5a receptor agonists, indicate that functional responses generated through the C5a receptor are not coordinately lost as the agonist potential of the peptides decreases. Rather, they are lost in a specific order: degranulation before Ca2+ flux, before chemotaxis (57). An alternative hypothesis is that Fc{gamma}R are coupled to multiple transduction pathways, associated with different functions. According to this explanation, different ligands should be able to activate Fc{gamma}R so that they can preferentially couple to a specific pathway. With regard to Fc{gamma}R, we speculate that signal intensities generated through these receptors as well as the induction of preferential specific transductional pathways might be dependent on the degree of receptor aggregation on the cell surface and/or the coaggregation of other receptors (i.e., CR3) rather than on the level of receptor occupancy.

Regarding CR3, it is now well established that this ß2 integrin can cooperate with Fc{gamma}R to mediate Ab-dependent effector functions. It was found that fibroblast transfectants expressing Fc{gamma}RIIIb alone or a tailless mutant of Fc{gamma}RIIa were unable to phagocyte IgG-coated erythrocytes. By contrast, transfectants that coexpressed any of these Fc{gamma}R and CR3 efficiently mediated phagocytosis (40, 42). Signaling through CR3 seems to play an important role not only in phagocytosis, but also in other responses triggered through Fc{gamma}R, such as stimulation of transient elevations in [Ca2+]i and activation of the respiratory burst by insoluble IC (58), as well as Ab-dependent cytotoxicity against tumor cells (45). On the other hand, there are a number of functions triggered through Fc{gamma}R, i.e., the initial adhesion and spreading of neutrophils on immobilized IC (43, 59) and the production of superoxide anion induced by either immobilized IC or IgG-coated latex beads, which do not involve the participation of CR3 (59). These findings suggest that the participation of CR3 in IgG-mediated functions is strongly dependent on the function itself as well as the properties of the IC employed as stimulus. Taking this into account and considering that CR3 is able to promote the apoptosis of activated neutrophils (22, 39), we analyzed whether CR3 was involved in the acceleration of apoptosis triggered by pIC and E-IgG. We found that acceleration of apoptosis was not modified by blocking Abs directed to either CD11b or CD18, suggesting that it does not involve a CR3-dependent pathway.

A link between Fc{gamma}R and apoptosis was first established in NK cells. Ortaldo et al. (60) showed that IL-2-activated NK cells undergo apoptosis as a consequence of treatment with anti-CD16 (anti-Fc{gamma}RIIIa) Abs or aggregated IgG. These results are in agreement with those of Azzoni et al. (61), who showed that the binding of anti-CD16 Abs or E-IgG to Fc{gamma}RIIIa induces apoptosis of IL-2-stimulated NK cells through a c-myc-dependent pathway. Eischen et al. (37), on the other hand, demonstrated that cross-linking of Fc{gamma}RIIIa induces the expression of FasL, which, in turn, facilitates both NK cell-mediated cytotoxicity and subsequent autocrine NK cell apoptosis. Consistent with our findings, these observations performed in NK cells also show contrasting effects induced by different ligands. While anti-CD16 mAbs or aggregated IgG trigger the apoptosis of IL-2-activated NK cells, Ab-coated tumor targets do not mediate any effect (61). Monomeric IgG, on the other hand, does not stimulate apoptosis of IL-2-treated NK cells, but, rather, it increases NK cell proliferation and up-regulates the expression of surface activation markers, cytotoxicity, cytokine production, and release of soluble IL-2R (62). Recent reports have revealed that Fc{gamma}R expressed by other hemopoietic cells may be also involved in the control of cell survival. The development of eosinophils in cultures of murine bone marrow is aborted as a consequence of Fc{gamma}R ligation through a CD32 (Fc{gamma}RII)- and Fas-dependent induction of apoptosis (38). Mature eosinophils also underwent apoptosis in response to Fc{gamma}RII engagement by anti-Fc{gamma}R Abs (38). In summary, these observations identify a new role for Fc{gamma}R: the regulation of cell survival. In vivo studies in Fc{gamma}R-deficient mice should be performed to define its physiologic significance.

Our results suggest that the stimulation of neutrophil apoptosis triggered by either pIC or E-IgG involves activation of the respiratory burst. Previous works have examined the impact of IRO on neutrophil apoptosis. Recently, Kasahara et al. (49), have shown that spontaneous cell apoptosis as well as the acceleration of apoptosis induced by anti-Fas Abs were partially prevented by catalase, supporting a role for H2O2 as a mediator of apoptosis. Consistent with this hypothesis, they also found that spontaneous in vitro cell death of CGD neutrophils was significantly decreased compared with that of normal neutrophils (49). Additional evidence was reported by Hannah and co-workers, who demonstrated that hypoxia causes a dramatic decrease in neutrophil apoptosis (63). Finally, other reports (33, 34, 39) show that after phagocytosis, neutrophils undergo apoptosis through an oxygen-dependent pathway.

It should be emphasized, however, that activation of the respiratory burst does not always result in stimulation of neutrophil apoptosis. In fact, sIC and aIgG (Fig. 1Go) as well as the calcium ionophore A23187 (47) induce the release of IRO but, at the same time, delay spontaneous apoptosis. Moreover, the chemotactic peptide FMLP (10-7 M), which is able to trigger a strong respiratory burst, does not stimulate neutrophil apoptosis (2, 3, 33) (R. Gamberale, unpublished observations). We speculate that the contrasting effects induced by different agonists on neutrophil apoptosis could be related at least in part to differences in the time pattern of the neutrophil respiratory response. The production of IRO triggered by sIC, aIgG, or FMLP (64) declines rapidly. By contrast, pIC, even when employed at very low concentrations (1 µg/ml), trigger a "respiratory marathon" that does not decline for up to 45 min after stimulation. This process may lead to the depletion of cellular antioxidant defenses and to an oxidative shift in the cellular redox state that could result in cell apoptosis.

Immune complexes play a critical role in the pathogenesis of several infectious and autoimmune diseases (65). They interact with Fc{gamma}R expressed by phagocytic cells and trigger the release of inflammatory cytokines, proteolytic enzymes, oxidative agents, and other toxic molecules. Our results support the idea that IC may also affect the course of inflammation by virtue of their ability to modulate neutrophil survival. In this regard, it is noteworthy that pIC, E-IgG, as well as immobilized IC markedly accelerate neutrophil apoptosis even in the presence of high concentrations of soluble IC (our unpublished observations). These observations suggest that promotion of apoptosis by tissue-deposited IC may represent a mechanism that favors the resolution of inflammation in either type II or III hypersensitivity reactions.


   Acknowledgments
 
We thank Fundación de la Hemofilia and Academia Nacional de Medicina for the use of the FACScan flow cytometer.


   Footnotes
 
1 This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires University School of Medicine, Fundación Alberto J. Roemmers, and Fundación Antorchas (Buenos Aires, Argentina). Back

2 Address correspondence and reprint requests to Dr. Romina Gamberale, Laboratorio de Immunología, Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 Buenos Aires, Argentina. Back

3 Abbreviations used in this paper: Fc{gamma}R, receptors for the constant region of immunoglobulin G; IC, immune complexes; pIC, precipitating immune complexes; E-IgG, mouse erythrocytes coated with specific immunoglobulin G antibodies; aIgG, human aggregated immunoglobulin G; sIC, soluble immune complexes; FasL, Fas ligand; MRBC, mouse erythrocytes; [Ca2+]i, intracellular Ca2+ concentration; URCL, relative chemiluminescence units; CL, chemiluminescence; SOD, superoxide dismutase; IRO, oxygen-reactive intermediates; CGD, chronic granulomatous disease. Back

Received for publication December 16, 1997. Accepted for publication May 27, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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