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1
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Laboratory of Immunology, Institute of Hematologic Research, National Academy of Medicine, and
Department of Microbiology, Laboratory of Immunogenetics, 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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to modulate human neutrophil
apoptosis. Neutrophils cultured with TNF- alone undergo a low but
significant increase in the number of apoptotic cells. More
interestingly, when neutrophils were pretreated with TNF- for 1–2
min at 37°C and then were exposed to a variety of agents such as
immobilized IgG, IgG-coated erythrocytes, complement-treated
erythrocytes, zymosan, PMA, zymosan-activated serum, fMLP,
Escherichia coli, and GM-CSF for 3 h at 37°C, a
marked stimulation of apoptosis was observed. Similar results were
obtained in neutrophils pretreated with TNF- for 30 min, 1 h,
3 h, and 18 h. Dose-dependent studies showed that TNF-
enhances neutrophil apoptosis at concentrations ranging from 1 to 100
ng/ml. In contrast to the observations made in neutrophils pretreated
with TNF-, there was no stimulation of apoptosis when TNF- was
added to neutrophils previously activated by conventional agonists.
Experiments performed to establish the mechanism through which TNF-
promotes neutrophil apoptosis showed that neither reactive oxygen
intermediates nor the Fas/Fas ligand system appear to be involved. Our
results suggest that TNF- plays a critical role in the control of
neutrophil survival by virtue of its ability to induce an apoptotic
death program which could be triggered by a variety of conventional
agonists. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. In vitro studies have identified a variety of agents that modulate neutrophil apoptosis. GM-CSF, IL-2, leukotriene B4, corticosteroids, and LPS inhibit neutrophil apoptosis (6, 7, 8, 9, 10, 11), whereas proteolytic enzymes, immune complexes, bacteria and virus induce neutrophil apoptosis (12, 13, 14, 15). Controversial results, on the other hand, have been reported regarding the effects of C5a, fMLP, G-CSF, and IL-6 (3, 5, 6, 7, 8, 9).
During the course of inflammatory processes, macrophages, lymphocytes,
and/or mast cells produce TNF-, a powerful priming agonist of
neutrophils (16, 17). Previous in vitro studies have shown
that TNF- enhances the expression of CD11b/CD18 on neutrophils,
increases neutrophil adhesion to endothelium, triggers adherent
neutrophils to release large amounts of reactive oxygen intermediates
(ROI), and promotes neutrophil degranulation, phagocytosis, and
Ab-dependent cell-mediated cytotoxicity (18, 19, 20, 21, 22).
TNF- has been variably reported either to induce, delay, or have no
effect on neutrophil apoptosis (6, 23, 24, 25). These
contrasting results could be explained, at least in part, by recent
findings of Murray et al. (26), who showed that although
prolonged incubation (>18 h) of human neutrophils with TNF- indeed
causes a decrease in the extent of apoptosis, TNF- can induce
apoptosis in a proportion of cells at earlier times (<8 h). In the
current work, we re-examine the impact of TNF- on neutrophil
survival. We found that TNF- plays a critical role in the control of
neutrophil survival. This function is related not only to the
recognized ability of TNF- to induce the apoptosis in a proportion
of neutrophils but also to its capacity, unique among other
inflammatory mediators, to induce an apoptotic death program which
could be rapidly triggered by a variety of conventional agonists.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following agents were used: human IgG, zymosan (Z), PMA,
fMLP, LPS (Escherichia coli 055:B5), Con A, acridine orange,
ethidium bromide, propidium iodide, catalase (from bovine liver, 50,000
U/mg protein) and superoxide dismutase (from bovine erythrocytes, 5,000
U/mg protein (Sigma, St. Louis, MO). Z-activated serum (ZAS), used as a
source of C5a, was prepared by incubating 15 mg Z with 1 ml fresh serum
with end-over-end rotation for 1 h at 37°C. Then serum was heat
inactivated for 30 min at 56°C. After spinning at 1000 x
g for 15 min at 4°C, the supernatant was collected and
stored at -70°C. IgG-coated erythrocytes (IgG-E) were prepared using
SRBC sensitized with subagglutinating amounts of rabbit IgG
anti-SRBC and suspended (1% v/v) in RPMI 1640 medium (Life
Technologies, Grand Island, NY) supplemented with 1% heat-inactivated
FCS (Life Technologies). Immobilized IgG (iIgG) was prepared by
incubating microplates (96-well flat-botton) with IgG (1 mg/ml in
saline) for 18 h at 37°C. Before use, plates were washed six
times with saline. Complement-treated erythrocytes (CE) were prepared
as follows. SRBC were sensitized with specific rabbit IgM Abs. Then
they were treated with C5-deficient mouse serum for 30 min at 37°C in
a shaking water bath. After washing, cells were suspended (1% v/v) in
RPMI 1640 medium supplemented with 1% FCS. Recombinant human TNF-,
IL-2, IL-4, IL-6, IL-12, and IFN-
were purchased from Sigma, and
IL-1, IL-8, IL-10, IL-18, and GM-CSF were purchased from R&D Systems
(Minneapolis, MN). Anti-human TNF- polyclonal neutralizing Ab was
purchased from R&D Systems.
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 it was drawn directly into heparinized plastic tubes.
Neutrophil isolation
Neutrophils were isolated by Ficoll-Hypaque gradient centrifugation (Ficoll; Pharmacia, Uppsala, Sweden; Hypaque, Winthrop Products, Buenos Aires, Argentina) and dextran sedimentation as described elsewhere (27). Contaminating erythrocytes were removed by hypotonic lysis. After washing, the cells (>96% of neutrophils on May-Grunwald-Giemsa-stained cytopreps) were resuspended in RPMI 1640 supplemented with 1% FCS.
Cell cultures
Aliquots of 0.10 ml of neutrophil suspensions (2.5 x
106/ml) were placed in 96-well flat-botton
microplates. Unless otherwise stated, neutrophils were treated with
TNF- (1–100 ng/ml) for 1–2 min at 37°C. Then cells were
stimulated with different agents. Apoptosis was evaluated after 3
h of culture at 37°C in 5% CO2, as described
below. In a separate set of experiments, the effect of TNF- on
neutrophil apoptosis was evaluated in whole-blood cultures. To this
aim, whole blood was diluted (20% v/v) in RPMI 1640 medium and
aliquots of 0.10 ml of this suspension were placed in 96-well
flat-botton microplates. Diluted blood samples were treated with
TNF- (10 ng/ml) for 1–2 min at 37°C. Then cells were stimulated
with different agents and apoptosis was evaluated after 3 h of
culture by fluorescence microscopy, as described below.
Quantitation of cellular apoptosis and viability by fluorescence microscopy
Quantitation was performed as previously described (28) 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 "structure"; variations in fluorescence intensity that reflect the distribution of euchromatin and heterochromatin. By contrast, apoptotic nuclei exhibit highly condensed chromatin that is uniformly stained by acridine orange. In fact, the entire apoptotic nucleus is present as bright spherical beads. To assess the percentage of cells showing morphologic features of apoptosis, at least 200 cells were scored in each experiment. Previous observations have demonstrated that morphological assessment of neutrophil apoptosis closely correlates with results obtained using other methods to assay apoptosis, such as propidium iodide staining and annexin V binding (29).
Quantitation of neutrophil apoptosis by annexin-V binding and flow cytometry
Annexin-V binding to neutrophils was performed using an
apoptosis detection kit (Immunotech, Marseille, France). Briefly,
2.5 x 105 neutrophils were incubated in the
presence or absence of TNF- (10 ng/ml) for 1–2 min, and then they
were treated with iIgG, ZAS, or IgG-E for an additional period of
3 h. After this period, cells were labeled with annexin-V FITC and
propidium iodide for 20 min at 4°C and analyzed by two-color flow
cytometry (FACScan flow cytometer; Becton Dickinson, San Jose, CA)
using CellQuest analysis software (Becton Dickinson), as previously
described (30). Results are reported as percentage of
annexin-V-positive cells.
Quantitation of neutrophil apoptosis by propidium iodide staining and flow cytometry
The proportion of neutrophils that display a hypodiploid DNA peak, i.e., apoptotic cells, was determined using a modification of the protocol of Nicoletti et al. (31). Briefly, cell pellets containing 2.5 x 106 neutrophils were suspended in 400 µl of hypotonic fluorochrome solution (propidium iodide, 50 µg/ml 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 in individual nuclei was measured using a FACScan flow cytometer (Becton Dickinson). 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 4–200.
Neutrophil shape change
Neutrophils (2.5 x 106/ml) suspended in RPMI 1640 medium with 1% FCS were incubated in a shaking water bath for 5 min at 37°C in the absence or presence of LPS (1 µg/ml) or Con A (10 µg/ml). Cells were then centrifuged, suspended in PBS, and fixed by the addition of an equal volume of 0.5% glutaraldehyde in PBS. Neutrophil shape change was measured by flow cytometry and results were expressed as mean forward scatter values.
CD18 expression
Neutrophils (2.5 x 106/ml) suspended in RPMI 1640 medium with 1% FCS were incubated in the absence or presence of LPS (1 µg/ml) or Con A (10 µg/ml) for 15 min at 37°C. Then cells were washed, fixed with 0.5% paraformaldehyde in PBS for 30 min at 4°C, and washed with PBS. Finally, cells were stained with FITC-conjugated anti-CD18 mAb. Fluorescence was recorded by flow cytometry. Results are expressed as the mean fluorescence intensity (MFI) in arbitrary fluorescence units.
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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In a first set of experiments, resting neutrophils were
preincubated with TNF- (10 ng/ml) for 1–2 min at 37°C and then
were cultured in the presence or absence of different stimuli. After
3 h of incubation at 37°C, apoptosis was revealed by
fluorescence microscopy using the fluorescent DNA-binding dye acridine
orange. Fig. 1
shows that when
neutrophils were cultured with TNF- alone, a low but significant
increase in the number of apoptotic cells was observed. By contrast,
when neutrophils were pretreated with TNF- for 1–2 min at 37°C
and then were cultured in the presence of iIgG, IgG-E, CE, PMA, ZAS, Z,
fMLP, E. coli, and GM-CSF, there was a marked increase in
the apoptotic rate. In contrast, no promotion of apoptosis was observed
using LPS, Con A, IL-1, IL-2, IL-8, IL-10, and IFN- as stimuli.
We then analyzed the specificity of action of TNF-. Using iIgG, Z,
IgG-E, and ZAS as apoptotic triggering agents, we observed that
preincubation of TNF- with a specific IgG polyclonal neutralizing Ab
abolished the proapoptotic effect of TNF- (data not shown).
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to induce apoptosis was then analyzed by flow cytometry using
FITC-labeled annexin-V, which specifically binds to PS, in combination
with propidium iodide staining. In agreement with the results described
above, we found that when neutrophils were pretreated with TNF- (10
ng/ml) for 1–2 min at 37°C and then were cultured in the presence of
iIgG, a marked increase in the number of apoptotic cells was observed
(Fig. 2). Comparable results were
observed using ZAS and IgG-E as triggering stimuli, being the
percentage of apoptotic cells of 5 ± 3, 4 ± 2, 3 ± 2,
9 ± 3, 37 ± 6, and 68 ± 5 for untreated cells and
cells treated with ZAS (1/10), iIgG, TNF- (10 ng/ml), TNF- plus
ZAS, and TNF- plus iIgG, respectively (mean ± SEM,
n = 5, p < 0.01, TNF- treated cells
vs TNF- + ZAS or TNF- + iIgG-treated cells). Similar results were
also observed when apoptosis was analyzed by propidium iodide staining
and flow cytometry. Treatment with TNF- enabled iIgG, ZAS, and IgG-E
to trigger a strong apoptotic response, as measured by the increased
proportion of neutrophils that display a hypodiploid DNA peak (Fig. 3).
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enhanced neutrophil apoptosis. To this aim, cells were cultured
for 1–2 min at 37°C with different concentrations of TNF-. Then
cells were exposed to ZAS or iIgG, and the percentage of apoptotic
neutrophils was assessed after 3 h of incubation either by
fluorescence microscopy or propidium iodide staining and flow
cytometry. Fig. 4 shows that pretreatment
with TNF- caused a dose-dependent stimulation of apoptosis at
concentrations ranging from 1 to 100 ng/ml.
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also showed that pretreatment with TNF-
was completely unable to increase apoptosis in neutrophils stimulated
with either LPS or Con A. To confirm that under our experimental
conditions LPS and Con A effectively triggered neutrophil activation,
their ability to increase the expression of CD18 as well as to
stimulate neutrophil shape change was evaluated by flow cytometry as
described in Materials and Methods. As expected, treatment
of neutrophils with either LPS (1 µg/ml) or Con A (10 µg/ml)
increased the expression of CD18 (MFI = 175 ± 23, 312
± 27, and 299 ± 16) and also induced neutrophil shape change
(MFSC = 312 ± 31, 487 ± 29, and 415 ± 21,
untreated, LPS, and Con A-treated neutrophils, respectively; mean
± SEM, p < 0.01, untreated vs LPS or Con A-treated
cells).
Previous observations suggest that standard preparative techniques
using 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 (32, 33). To rule
out the possibility that our results could be related to the induction
of cell injury or "priming" during purification, we performed
experiments in whole-blood cultures as described in Materials and
Methods. Diluted whole-blood samples (20% v/v in RPMI 1640
medium) were incubated with TNF- (10 ng/ml) for 1–2 min at 37°C
and then they were exposed to different agents. After 3 h of
incubation at 37°C, apoptosis was revealed by fluorescence
microscopy. Fig. 5 shows that, in
agreement with the observations made in purified neutrophils (see Fig. 1), pretreatment of whole-blood samples with TNF- induced a marked
stimulation of apoptosis in neutrophils stimulated by either IgG-E, CE,
PMA, ZAS, and Z.
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The above-described experiments were all performed with
neutrophils pretreated with TNF- for 1–2 min at 37°C. To analyze
whether the proapoptotic effect of TNF- was also observed with
longer pretreatments, neutrophils were cultured for different times
with TNF- and then were stimulated by iIgG. Fig. 6 shows that TNF- markedly increased
the proportion of apoptotic cells at all of the times assessed.
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was reversible. To this
end, we performed another set of experiments in which TNF- was
removed from the culture medium before the addition of the triggering
stimulus. Neutrophils were cultured with TNF- for 2 min at 37°C,
washed three times, and resuspended in culture medium. Cells were
exposed to Z (50 µg/ml) for an additional period of 3 h at
37°C, and apoptosis was then revealed by fluorescence microscopy. We
found that removal of TNF- from the culture medium did not impair
its ability to promote apoptosis: percent apoptosis = 3 ± 2,
4 ± 2, 11 ± 2, and 61 ± 7, for untreated cells and
cells treated with Z (50 µg/ml), TNF- (10 ng/ml), and Z + TNF-
(mean ± SEM, n = 6, p < 0.05,
untreated vs TNF--treated cells, and p < 0.01,
TNF- vs Z + TNF--treated cells).
The observations described above were made in experiments in which
resting neutrophils were pretreated with TNF- for different times
before the addition of triggering stimuli. We then analyzed whether
neutrophils first stimulated with iIgG, IgG-E, ZAS, or GM-CSF undergo
an increase in their apoptotic rate as a consequence of the addition of
TNF- to the culture medium. Fig. 7
shows that, in contrast to the observations made in TNF--pretreated
neutrophils, we observed no stimulation of apoptosis when TNF- was
added to activated neutrophils. These results could be explained, at
least in part, considering previous findings which indicated that
neutrophil activation by different stimuli induces the shedding of
TNF- receptors (34, 35). In agreement with these
reports, we observed that neutrophil activation by iIgG, IgG-E, ZAS, or
GM-CSF markedly decreased the expression of both TNF- receptors
(TNF-R55 and TNF-R75) from the cell surface (data not shown).
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does not appear to involve the
Fas/Fas ligand (FasL) system nor the production of ROI
Neutrophils express not only Fas but also FasL. The Fas/FasL
system appears to be responsible, at least in part, for the rapid rate
of spontaneous apoptosis observed for unstimulated neutrophils
(36). We next examined whether promotion of apoptosis by
TNF- was associated with an increase in the expression of Fas and/or
FasL. Neutrophils were pretreated for 1–2 min at 37°C with or
without TNF- and then they were cultured for 2 h with iIgG.
Using the mAb CH-11 directed to Fas and the mAb G247–4 directed to
FasL, we found no differences in the expression of Fas or FasL between
untreated and TNF--treated cells. The MFI for Fas expression in
control cells and cells treated with iIgG, TNF- (10 ng/ml), and
TNF- plus iIgG was 28 ± 5, 29 ± 4, 24 ± 5, and
23 ± 5, respectively (mean ± SEM, n = 5).
FasL expressión in cells treated as described above was 7 ±
3, 8 ± 2, 6 ± 3, and 8 ± 3, respectively (mean
± SEM, n = 5). These results suggest that the Fas/FasL
system is not involved in the acceleration of apoptosis induced by
TNF-. This conclusion was further supported by the fact that
pretreatment of neutrophils with a blocking mAb directed to FasL
(NOK-1), used at concentrations 5-fold higher than those needed to
saturate all binding sites as determined by FACS analysis, did not
impair the acceleration of apoptosis induced by TNF-: percent
apoptosis after 3 h of culture, 74 ± 11 vs 68 ± 7 for
TNF--treated neutrophils incubated with iIgG in the absence or
presence of the mAb NOK-1, respectively (n =
3).
Previous work has shown that after activation by different stimuli,
neutrophils undergo apoptosis through an oxygen-dependent pathway
(13, 14, 37, 38). To analyze whether ROI were involved in
the acceleration of apoptosis induced by TNF-, we determined the
effect of catalase and superoxide dismutase (SOD). Fig. 8 shows that neither catalase nor SOD
impaired the acceleration of apoptosis induced by TNF- in cells
incubated for 3 h with either IgG-E or Z. These data contrast with
the results obtained in neutrophils cultured for 18 h only with
IgG-E in which, as we previously described (14), the
promotion of apoptosis induced by IgG-E was not modified by SOD (data
not shown) but was almost completely abrogated by catalase: percent
apoptosis = 33 ± 6, 28 ± 5, 69 ± 11, and 34
± 4, for untreated cells and cells treated with catalase (200 U/ml),
IgG-E (0.25%), and IgG-E plus catalase, respectively (mean ±
SEM, n = 5).
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, assays were performed
using neutrophils isolated from three patients with chronic
granulomatous disease (CGD), a rare hereditary disorder characterized
by a diminished or absent production of ROI due to a defect in any one
of the components of NADPH oxidase (39). Neutrophils from
these patients produced, in response to fMLP or Z,
O2- levels <1% those produced
by normal neutrophils (data not shown). Our results showed that, as
observed with normal neutrophils, there was a strong stimulation of
apoptosis in CGD neutrophils cultured with TNF- (2 min at
37°C) and then exposed to Z: percent apoptosis revealed at 3 h
of culture by fluorescence microscopy = 4 ± 2, 11 ± 6,
2 ± 1, and 53 ± 8 for untreated cells and cells treated
with TNF- (10 ng/ml), Z (50 µg/ml), and TNF- + Z,
respectively (n = 3). These results suggest that
promotion of neutrophil apoptosis by TNF- does not involve an
oxygen-dependent pathway.
Promotion of neutrophil apoptosis appears to be a selective action
of TNF-
We next analyzed whether other cytokines could reproduce the
effects of TNF-. The following cytokines were assessed: IL-1,
IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, IFN-, and GM-CSF.
Neutrophils were incubated with each of these cytokines for 1–2 min at
37°C and then were treated with Z or IgG-E. Apoptosis was revealed by
fluorescence microscopy after 3 h of culture. Fig. 9 shows that in contrast to the
observations made in TNF--treated neutrophils, none of these
cytokines was able to increase apoptosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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plays a critical
role in the control of neutrophil survival by inducing an apoptotic
death program which can be rapidly triggered by a variety of stimuli.
Thus, our data reveal that when neutrophils were pretreated with
TNF- and then were exposed to different inflammatory agents, there
was a marked stimulation of apoptosis (range, 30–75%). By contrast,
in agreement with previous reports (23, 24, 25, 26), we found a
slight increase in the number of apoptotic cells (range, 9–18%) when
neutrophils were cultured only with TNF-.
Using a broad panel of stimuli which includes immune complexes (IgG-E,
iIgG), physiologic peptides (fMLP), bacteria (E. coli),
yeast (Z from Saccharomyces cerevisiae), pharmacologic
agents that activate protein kinase C (PMA), cytokines (IL-1, IL-2,
IL-8, IL-10, IFN-, and GM-CSF), and ZAS (used as a source of C5a),
we found marked differences in their ability to trigger apoptosis of
neutrophils treated with TNF-. On the basis of these differences,
these stimuli should be classified in three different groups: 1) potent
inducers of apoptosis (range, 55–75%) such as IgG-E, iIgG, E.
coli, and Z; 2) moderate and low inducers of apoptosis (range,
30–45%), which include fMLP, ZAS, CE, PMA, and GM-CSF; and 3) agents
unable to enhance apoptosis, such as LPS, Con A, IL-1, IL-2, IL-8,
IL-10, and IFN-. Not only conventional agonists appear to be able to
accelerate apoptosis of TNF- primed neutrophils. Recent results
published by Kettritz et al. (40) showed that
extracellular matrix proteins, such as fibronectin, collagen I,
collagen IV, and laminin, may also be able to induce a low but
significant increase in the apoptotic rate of TNF--treated
neutrophils.
The mechanisms through which TNF- increases neutrophil apoptosis
have not been defined yet. The observation that a blocking Ab directed
to FasL was unable to prevent apoptosis supports the notion that the
Fas/FasL system is not involved. Moreover, the inability of catalase
and SOD to decrease apoptosis and, more importantly, the fact that
TNF- was able to promote apoptosis of CGD neutrophils argues against
the participation of an oxidative-dependent pathway. In this regard, it
is important to consider our previous results indicating that 1) IgG-E
stimulates apoptosis of resting neutrophils, revealed at 12–18 h of
culture (this effect was not evident in the current study since
apoptosis was always revealed at 3 h of culture); and 2) the
enhancement in the apoptotic rate induced by IgG-E is almost completely
abrogated by catalase, and it was not observed in neutrophils from CGD
patients (14). These data suggest that, in contrast to
what was observed in TNF--primed neutrophils, stimulation of
apoptosis of resting neutrophils by IgG-E involves an oxygen-dependent
pathway. Similar results were obtained by using iIgG and Z as stimulus.
Catalase markedly inhibited apoptosis of resting neutrophils induced by
iIgG or Z (revealed at 18 h of incubation), whereas it did not
exert any effect on apoptosis of TNF--primed neutrophils triggered
by these stimuli (G. Salamone, unpublished results). Together, these
data suggest that TNF- not only enables different stimuli to trigger
a strong apoptotic response, but also modifies the mechanisms through
which these stimuli induce neutrophil apoptosis.
We have also observed that TNF- does not increase apoptosis of
neutrophils first activated by conventional agonists. This result could
be explained, at least in part, considering previous findings from
Porteu and Nathan (34) which indicated that neutrophil
activation by a variety of agents results in the shedding of TNF-R from
the cell surface. In agreement with these results, we observed that
activation of neutrophils by iIgG, IgG-E, ZAS, or GM-CSF results in a
rapid loss of both TNF- receptors (TNF-R55 and TNF-R75; our
unpublished results).
Having demonstrated that TNF- promotes neutrophil apoptosis, we also
sought to determine whether other cytokines such as IL-1, IL-2,
IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, GM-CSF, and IFN- could also
exert a similar function. Using Z and IgG-E as triggering stimuli, we
found no enhancing effects, suggesting that TNF- has the unique
ability, not shared by other cytokines, to induce an apoptotic death
program which could be rapidly triggered by a variety of conventional
agonists.
Although our results support a critical role for TNF- in promoting
neutrophil apoptosis, additional experiments are required to establish
the relevance of this regulatory mechanism. In this regard, however, it
is important to consider recent findings published by Skerrett et al.
(41), who examined the role of TNF-R in lung acute
inflammation. They found that neutrophil infiltration in the lungs
after exposure to aerosolized Pseudomonas aeruginosa, but
not after exposure to aerosolized LPS, was augmented in mice lacking
TNF-R, in comparison with wild-type animals, despite diminished
bronchoalveolar concentrations of chemokines. Since exposure to TNF-
enables Gram-negative bacteria, but not LPS, to trigger a strong
apoptotic response, these observations could reflect the ability of
P. aeruginosa-elicited, but not LPS-elicited neutrophils, to
survive longer in lung airspaces from TNF-R-deficient animals.
In summary, we found that TNF- plays an important role in the
control of neutrophil survival by virtue of its ability to induce an
apoptotic death program that can be triggered by a variety of
conventional agonists. Additional experiments are required to elucidate
the signaling pathways involved in TNF- priming as well as to define
the impact of this mechanism in the resolution of acute inflammatory
processes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gabriela Salamone, Laboratorio de Inmunología, IIHEMA, Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 Buenos Aires, Argentina.
3 Abbreviations used in this paper: PS, phosphatidylserine; iIgG, immovilized human IgG; IgG-E, IgG-coated erythrocytes; CE, complement-treated erythrocytes; Z, zymosan; ZAS, Z-activated serum; ROI, reactive oxygen intermediate; MFSC, mean forward scatter; MFI, mean fluorescence intensity; SOD, superoxide dismutase; CGD, chronic granulomatous disease.
Received for publication September 14, 2000. Accepted for publication December 18, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 M. Hu, X. Lin, Q. Du, E. J. Miller, P. Wang, and H. H. Simms Regulation of polymorphonuclear leukocyte apoptosis: role of lung endothelium-epithelium bilayer transmigration Am J Physiol Lung Cell Mol Physiol, February 1, 2005; 288(2): L266 - L274. [Abstract] [Full Text] [PDF]
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C.-Y. Liu, A. Takemasa, W. C. Liles, R. B. Goodman, M. Jonas, H. Rosen, E. Chi, R. K. Winn, J. M. Harlan, and P. I. Chuang Broad-spectrum caspase inhibition paradoxically augments cell death in TNF-alpha -stimulated neutrophils Blood, January 1, 2003; 101(1): 295 - 304. [Abstract] [Full Text] [PDF]
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R. J. Coakley, C. Taggart, N. G. McElvaney, and S. J. O'Neill Cytosolic pH and the inflammatory microenvironment modulate cell death in human neutrophils after phagocytosis Blood, October 16, 2002; 100(9): 3383 - 3391. [Abstract] [Full Text] [PDF]
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J. Schettini, G. Salamone, A. Trevani, S. Raiden, R. Gamberale, M. Vermeulen, M. Giordano, and J. R. Geffner Stimulation of neutrophil apoptosis by immobilized IgA J. Leukoc. Biol., October 1, 2002; 72(4): 685 - 691. [Abstract] [Full Text] [PDF]
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A. S. Cowburn, K. A. Cadwallader, B. J. Reed, N. Farahi, and E. R. Chilvers Role of PI3-kinase-dependent Bad phosphorylation and altered transcription in cytokine-mediated neutrophil survival Blood, September 18, 2002; 100(7): 2607 - 2616. [Abstract] [Full Text] [PDF]
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 K. O. Kisich, M. Higgins, G. Diamond, and L. Heifets Tumor Necrosis Factor Alpha Stimulates Killing of Mycobacterium tuberculosis by Human Neutrophils Infect. Immun., August 1, 2002; 70(8): 4591 - 4599. [Abstract] [Full Text] [PDF]
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S. H. Gregory and E. J. Wing Neutrophil-Kupffer cell interaction: a critical component of host defenses to systemic bacterial infections J. Leukoc. Biol., August 1, 2002; 72(2): 239 - 248. [Abstract] [Full Text] [PDF]
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 C. A. Pettersen and K. B. Adler Airways Inflammation and COPD* : Epithelial-Neutrophil Interactions Chest, May 1, 2002; 121 (2009): 142S - 150S. [Abstract] [Full Text] [PDF]
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 R. Kooijman, A. Coppens, and E. Hooghe-Peters IGF-I Inhibits Spontaneous Apoptosis in Human Granulocytes Endocrinology, April 1, 2002; 143(4): 1206 - 1212. [Abstract] [Full Text] [PDF]
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