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*
Laboratory of Immunology, Institute of Hematologic Research, National Academy of Medicine, and
Department of Microbiology, Buenos Aires University School of Medicine, Buenos Aires, Argentina
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor
type II (FcRII), but not to FcRIII, markedly diminished the
acceleration of apoptosis triggered by either pIC or E-IgG, supporting
a critical role for FcRII 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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Receptors for the constant region of IgG
(FcR)3 fall into three major classes: FcRI
(CD64), FcRII (CD32), and FcRIII (CD16). They comprise nine
membrane-associated and three soluble FcR molecules, produced by
alternative splicing of FcR transcripts or by proteolysis of surface
receptors. Most membrane-associated FcR exist as
hetero-oligomeric complexes with a ligand-binding 
-chain, which
determines affinity and isotype specificity, and a signaling component
comprising -, ß-, or 
-chains (10, 11, 12, 13).
Human neutrophils constitutively express two low affinity FcR:
FcRIIa (10,000–20,000 sites/cell) and FcRIIIb (100,000–200,000
sites/cell). FcRIIa is a transmembrane molecule, while FcRIIIb 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 FcR 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 FcR aggregation
(16, 17, 18, 19). To our knowledge, no previous work has examined the effect of
FcR engagement on neutrophil survival.
In the present study we analyze whether the activation of FcR 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 FcRIII, and IV.3
(IgG2b), which recognizes human FcRII, 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immune complexes constitute a heterogeneous group of FcR
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 FcR 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 1
, 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. 1).
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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). 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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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
FcRIIa and does not require IC phagocytosis
The roles of FcRIIa and FcRIIIb in the stimulation of
apoptosis by pIC and E-IgG were evaluated using blocking mAb directed
to FcRIIa (IV.3) and FcRIIIb (3G8). Incubation of neutrophils
with these mAb had no effect on spontaneous apoptosis. As shown in
Figure 3, 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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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 FcR 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 FcR 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. 4). 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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Recently, Walzog et al. (22) have shown that cross-linking of CR3
(CD11b/CD18, Mac-1, mß2) promotes
apoptosis in neutrophils that have been activated by either treatment
with TNF- 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 FcR 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 5 show that these mAb did not prevent the
acceleration of apoptosis induced by pIC or E-IgG.
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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 6, both stimuli induced similar
fluctuations in intracellular Ca2+ concentrations.
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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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A role for IRO in the acceleration of apoptosis triggered by pIC
was examined using catalase and SOD. The results presented in Figure 8 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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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. 9). 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 FcR.
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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
FcR 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 FcR ligands on neutrophil apoptosis are
consistent with these observations. Moreover, we found differences
between particulate (pIC and E-IgG) and soluble (sIC and aIgG) FcR
ligands with regard to other neutrophil functions. Thus, particulate
and soluble FcR ligands were equally effective to trigger both an
increase in intracellular Ca2+ concentrations (Fig. 5
) 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-
synthesis (not shown). A possible explanation for these contrasting
effects is that different signal intensities might be required to
induce distinct FcR-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 FcR are coupled to multiple transduction
pathways, associated with different functions. According to this
explanation, different ligands should be able to activate FcR so
that they can preferentially couple to a specific pathway. With regard
to FcR, 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 FcR to mediate Ab-dependent effector
functions. It was found that fibroblast transfectants expressing
FcRIIIb alone or a tailless mutant of FcRIIa were unable to
phagocyte IgG-coated erythrocytes. By contrast, transfectants that
coexpressed any of these FcR 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 FcR, 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 FcR, 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 FcR 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-FcRIIIa) 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 FcRIIIa 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 FcRIIIa 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 FcR 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 FcR ligation through a CD32
(FcRII)- and Fas-dependent induction of apoptosis (38). Mature
eosinophils also underwent apoptosis in response to FcRII engagement
by anti-FcR Abs (38). In summary, these observations identify a
new role for FcR: the regulation of cell survival. In vivo studies
in FcR-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. 1) 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 FcR
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.
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Acknowledgments |
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Footnotes |
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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.
3 Abbreviations used in this paper: FcR, 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.
Received for publication December 16, 1997. Accepted for publication May 27, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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