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on apoptosis of human neutrophils
* Central Laboratory of the Netherlands Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam; and
Departments of Pediatrics and
Pathology, Academic Medical Center, University of Amsterdam, The Netherlands
Correspondence: Taco W. Kuijpers, M.D., Ph.D., CLB, Dept. IHE, F116, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. E-mail: T_Kuijpers{at}CLB.NL
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) was reported by some to have
pro-apoptotic and by others to have anti-apoptotic effects on
neutrophils. The aim of this study was to explain these contradictory
results. We found that TNF- at low concentrations strongly decreased
apoptosis of neutrophils. However, at higher concentrations, TNF-
lost its protective effects, and also reversed the protective effects
of interferon-
(IFN-) and granulocyte-macrophage
colony-stimulating factor (GM-CSF). This pro-apoptotic effect of
TNF- was blocked by anti-CD11b and was absent in neutrophils from
patients with chronic granulomatous disease, which cannot produce toxic
oxygen metabolites. Under these circumstances, we found that TNF-
retained its anti-apoptotic effects even at high concentrations. In
conclusion, the protective effects against apoptosis of IFN-,
GM-CSF, and TNF- itself are overruled when the concentration of
TNF- is high enough to produce a respiratory burst. These dual,
concentration-dependent effects of TNF- provide an explanation for
previous controversial reports and support a dominant role for TNF-
in neutrophil apoptosis.
Key Words: leukocyte adhesion deficiency • chronic granulomatous disease • granulocyte-macrophage colony-stimulating factor • interferon- • CD11b/CD18
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Neutrophils are short-lived cells. It is important that the massive cell death of infiltrated neutrophils occurs in a controlled way, with minimal release of the hazardous contents. This regulated cell death is essential to limit the inflammatory response and allows repair to take place. Apoptosis, or programmed cell death, is therefore a key mechanism to control the duration of the early inflammatory response as well as tissue damage caused by neutrophils [3 ].
Apoptosis and necrosis, the two pathways of cell death, differ in various ways. Necrosis is characterized by swelling of the cell, rupture of the cell membrane, and uncontrolled release of the cellular contents into the surroundings. In contrast, during apoptosis, the plasma membrane remains intact. Apoptotic cells are phagocytosed and degraded by macrophages, and are thus safely removed from the tissues [4 ].
The cellular machinery that controls apoptosis has been studied extensively in numerous cell types over the past few years. Membrane proteins such as the tumor necrosis factor (TNF) receptors and Fas, intracellular proteins of the Bcl-2 family, and activation by enzymatic cleavage of caspases can either induce or reverse a death signal from the cell membrane to the nucleus [5 6 7 ]. A host of extracellular stimuli with pro-apoptotic or anti-apoptotic stimuli has been identified.
Compared with other cell types, the pathways that lead to apoptosis of
neutrophils have been studied less extensively, and controversial
results have been reported. In general, it seems that pro-inflammatory
cytokines protect neutrophils from apoptosis [8
,
9
], but for TNF- protective as well as
apoptosis-inducing activity has been reported [10
,
11
]. The ability of this cytokine to induce cell death
was appreciated when it was first characterized. More recently, it was
found that in some cases TNF- can protect cells from apoptosis,
through pathways that are linked to activation of the transcription
factor NF-
B [12
].
Outside-in signaling through adhesion of cells to other cells or extracellular matrix (ECM) proteins is another mechanism that can have a profound effect on apoptosis [13 , 14 ]. Indeed, for most cultured cells, but also for leukocytes closely related to neutrophils, such as eosinophilic granulocytes, adhesion is essential for cell survival [15 ]. In contrast, adhesion seems to be a pro-apoptotic signal in neutrophils: binding via the integrin CD11b/CD18 has been linked to reduced survival [16 ].
In vivo, the migrated neutrophil is surrounded by a wide variety of ECM proteins, and is subject to the actions of numerous inflammatory cytokines. It is likely that the sum of these stimuli controls cell activation and cell death.
To determine the cause for the controversial reports on the effects of
TNF- on neutrophil apoptosis, we investigated the role of TNF- in
comparison to two known inhibitors of neutrophil apoptosis,
interferon- (IFN-) and granulocyte-macrophage colony-stimulating
factor (GM-CSF). Furthermore, we studied whether involvement of
CD11b/CD18 and production of oxygen metabolites were important in
mediating the effects of these three cytokines.
We found, in general, that TNF-, IFN-, and GM-CSF protect
neutrophils from apoptosis. This process was dependent on de
novo protein synthesis. However, at high doses, TNF- no longer
protected against apoptosis, and in addition potently inhibited
anti-apoptotic effects of IFN- and GM-CSF. This pro-apoptotic
pathway was absent if CD11b on neutrophils from healthy donors was
blocked by antibodies, and in cells of patients suffering from
leukocyte adhesion deficiency-1 (LAD-1) or LAD-1-variant patients,
which do not express CD18 or lack functional CD18, respectively. The
pro-apoptotic effect of high doses of TNF- was also absent in cells
from patients with chronic granulomatous disease (CGD), which cannot
produce toxic oxygen metabolites. Under these circumstances, we found
that TNF- retained its anti-apoptotic effects even at high
concentrations.
Thus, our results provide a comprehensive and elegant explanation for
the controversial reports on TNF-, showing that TNF- has opposing
effects on neutrophil apoptosis, depending on the concentration and
involvement of the integrin CD11b/CD18. The essential process that
shifts the balance toward apoptosis seems to be the occurrence of a
respiratory burst, which overrules other anti-apoptotic stimuli.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was purchased from Calbiochem (La Jolla, CA),
GM-CSF from SanverTECH (Heerhugowaard, The Netherlands), IFN- from
Boehringer Mannheim (Mannheim, Germany). Propidium iodide (PI) and
cycloheximide were purchased from Sigma Chemical (St. Louis, MO) and
bovine serum albumin (BSA) from Organon Teknika (Boxtel, The
Netherlands). Annexin-V-fluorescein isothiocyanate (FITC) was purchased
from Bender MedSystems (Vienna, Austria). All other reagents, if not
otherwise stated, were purchased from Merck (Darmstadt, Germany).
Isolation and treatment of granulocytes
Granulocytes were isolated from heparinized blood of healthy
donors as described [17
]. Briefly, 20 mL of blood was
further anticoagulated and diluted with 20 mL of 10% trisodium
citrate/phosphate-buffered saline (PBS). Mononuclear cells and
platelets were removed by density gradient centrifugation over isotonic
Percoll (Pharmacia, Uppsala, Sweden) with a specific gravity of 1.076
g/mL. Erythrocytes were lysed by short treatment of the pellet fraction
with ice-cold isotonic NH4Cl solution (155 mM
NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.4).
The remaining granulocytes were washed once in PBS and then resuspended in HEPES medium [20 mM HEPES, 132 mM NaCl, 6 mM KCl, 1 mM CaCl2, 1 mM MgSO4, 42 mM K2HPO4 3H2O, 5.5 mM glucose and 0.4 % (wt/vol) HSA, pH 7.4].
Light microscopic evaluation of May Grünwald/Giemsa-stained cytospins of the granulocyte fraction revealed a purity of more than 99%, with more than 95% neutrophils.
Apoptosis assay
The percentage of apoptotic cells was determined as described
before [8
]. Briefly, the neutrophils were washed once in
HEPES and then resuspended at a concentration of 2 x
106 cells/mL in Iscove’s modified Dulbecco’s medium
(IMDM; BioWhittaker, Brussels, Belgium) supplemented with 10%
heat-inactivated FCS (GIBCO-BRL, Paisley, UK), penicillin (100 IU/mL,
Yamanouchi, Tokyo, Japan), streptomycin (100 µg/mL, GIBCO-BRL), and
glutamine (300 µg/mL). On 96-well plates (NUNC Brand Products,
Roskilde, Denmark), aliquots of 100 µL of cell suspension were
pre-incubated with monoclonal antibodies (15 min), then stimulated with
cytokines and subsequently maintained overnight (17 h) in a 5%
CO2 incubator at standard conditions. In experiments with
combinations of cytokines, the cells were preincubated for 15 min with
IFN- or GM-CSF, followed by addition of TNF-. In some control
experiments, the order of addition was reversed, which resulted in
similar findings.
After this overnight incubation, the PMN were treated with EDTA/PBS (5 mM, 30 min, 37°C) to release adherent cells from the wells. The cells were then taken from the wells and washed once in ice-cold HEPES medium with additional Ca2+ (2.5 mM). All further steps were performed in this medium. The cells were then incubated with Annexin-V-FITC (1:500), which specifically binds phosphatidylserine residues on the cell membrane of apoptotic cells. After 45 min, the cells were washed once and stained with PI (5 µg/mL), a fluorescent dye that will bind to DNA once the cell membrane has become permeable. The cells were then immediately analyzed with a FACScan (Becton Dickinson, San Jose, CA). Under all conditions tested, percentages of surviving cells (Annexin-Vneg, PIneg) were compared. Necrosis (Annexin-Vneg, PIpos) was minimal (<3%) under all circumstances.
FACS analysis
After isolation, granulocytes were incubated in HEPES medium (1
mM Ca2+) at a concentration of 107 cells/mL,
stimulated with cytokines for 30 min at 37°C, then washed in ice-cold
HEPES medium. The cells were incubated with mouse monoclonal antibodies
(5 µg/mL) for 1 h at 4°C, washed once, and re-incubated with
R-phycoerythrin (RPE)-labeled goat-anti-mouse-Ig antibodies (10
µg/mL; DAKO, Glostrup, Denmark). After washing, the binding of
antibodies was assessed by measuring the mean fluorescence intensity by
FACS analysis, and the results were compared to fluorescence levels of
isotype controls.
Statistics
Student’s t test for paired samples (two-tailed) was
used for statistical analysis. Student’s t test for
independent samples was used where indicated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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on survival of neutrophils has been described to either induce or
decrease apoptosis of neutrophils. We found that the effects of TNF-
were dose dependent: at low doses (0.1–1 ng/mL) survival increased,
but at higher doses (10 ng/mL) this effect was lost (Fig. 1A
). The anti-apoptotic dose of TNF- correlated with the plasma
levels found in human volunteers after endotoxin administration (0.3
ng/mL compared with 0.03 ng/mL in controls) [18
]. In
later experiments, we found that a 10-fold higher concentration of
TNF- (100 ng/mL) had a similar effect (see Fig. 5). Because TNF-
is known to up-regulate the integrin CD11b/CD18 on neutrophils, we
studied the effect of Fab fragments of 44a, a blocking mAb to CD11b. In
the absence of TNF-, there was no effect of anti-CD11b Fab. However,
at high doses of TNF- (as tested up to 100 ng/mL), the pro-apoptotic
effect was blocked completely by these Fab fragments. Isotype-matched
control antibodies had no effect (not shown). Thus, with CD11b blocked,
it was clear that the anti-apoptotic effect of low doses of TNF- was
also present at these higher concentrations (Fig. 1A)
. Identical
results were seen when blocking Fab fragments of mAb IB4 to the CD18
(ß2)-integrin subunit were used (not shown). In contrast,
the increased neutrophil survival induced by two other pro-inflammatory
cytokines, IFN- and GM-CSF, was maintained over a wide concentration
range. The 44a Fab fragments had no effect on IFN--induced
GM-CSF-induced survival (Fig. 1B
and 1C)
.
|
we studied cells from patients suffering from
a disease termed LAD-1/variant, characterized by normal expression but
lack of functional activity of CD18 (ß2) integrins
[19
]. In cells from two nonrelated patients, high-dose
TNF- was ineffective in inducing apoptosis, and addition of 44a Fab
had no effect (Fig. 2
). The effects of IFN- (Fig. 2)
or GM-CSF (not shown) were not
different from those observed with neutrophils from normal individuals.
In neutrophils from two classic LAD-1 patients, on which
ß2 integrins are not expressed at all, we found the same
results (not shown).
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, IFN-, and GM-CSF on expression and
activation of CD11b-induced apoptosis, we studied expression levels
and activation of CD11b after stimulation with the three cytokines of
interest. After stimulation with TNF- we found a dose-dependent
increase in CD11b expression (Fig. 3A
) and an increase in the expression of an activation epitope of
CD11b that is specifically recognized by mAb CBRM1/5
[20
] (Fig. 3B)
. The expression of the activation epitope
also increased relative to CD11b expression itself, suggesting that the
percentage of activated CD11b molecules on the cell membrane increased
(Fig. 3A
and 3B)
. As with TNF-, treatment with GM-CSF resulted in
an increase in expression of CD11b and its activation epitope (Fig. 3A
and 3B)
. In contrast, IFN- did not alter the expression levels or
activation of CD11b (Fig. 3A
and 3B)
. Because GM-CSF was as potent as
TNF- in up-regulating and activating CD11b, it seems that besides
CD11b activation an additional signal provided by TNF- but not by
GM-CSF is necessary to drive the cells toward apoptosis.
|
-induced neutrophil
apoptosis did not result in a
decrease in cell survival (Fig. 4
), and treatment with 44a Fab had no additional protective effect
(not shown). This shows that if a respiratory burst does not take
place, TNF- retains its protective effects. The effects of IFN-
or GM-CSF treatment were not different from those observed with
neutrophils from normal individuals.
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and GM-CSF on TNF--induced apoptosis of
neutrophils and GM-CSF had protective effects against
spontaneous apoptosis of neutrophils, we tested whether treatment with
these cytokines could also protect neutrophils from apoptosis induced
by high-dose TNF-. Whereas IFN- enhanced survival when the cells
had been treated with no or low doses of TNF- (0–0.1 ng/mL), at
higher doses of TNF- addition of IFN- had no effect (Fig. 5A
). In contrast, GM-CSF even significantly enhanced the
pro-apoptotic effects of high-dose TNF- (Fig. 5B)
.
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, GM-CSF, and
TNF- itself.
Protein synthesis in prevention of apoptosis by TNF-, IFN-,
and GM-CSF
To study whether the cellular machinery necessary for the
increased survival of neutrophils by the three cytokines is present or
has to be newly synthesized, we treated the cells with cycloheximide
(CHX). This cytotoxic drug inhibits protein synthesis. The
concentration of CHX that we used (1 µg/mL) had no effect on the
basal percentage of apoptosis of neutrophils after overnight
incubation, but was sufficient to completely block de novo
synthesis of the FcRI, which normally occurs after overnight
incubation with IFN- [8
] (data not shown). The
increase in neutrophil survival induced by all three cytokines was
blocked by 1 µg/mL CHX (Fig. 6A-C
), suggesting that de novo protein
synthesis is necessary for the induction of the protective effects of
these three cytokines.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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has two opposite effects on neutrophil
apoptosis, and thus provide an explanation for previous contradictory
reports. First, TNF- prevents apoptosis over a wide concentration
range. This effect is dependent on protein synthesis and is similar to
the effects of treatment with other pro-inflammatory cytokines such as
IFN- and GM-CSF. Second, at higher concentrations of TNF-
(10–100 ng/mL), a dominant pro-apoptotic effect becomes apparent, a
process mediated by the production of reactive oxygen species and
dependent on the activation of CD11b/CD18. This was illustrated by the
absence of the pro-apoptotic effect of TNF- in neutrophils of
patients with congenital defects in the expression or function of
CD11b/CD18 molecules (i.e., LAD-1 and LAD-1/variant) or defects in the
production of toxic oxygen metabolites (i.e., CGD).
It is known that treatment with TNF- leads to the consecutive
up-regulation and activation of the integrin CD11b/CD18 on neutrophils.
If CD11b/CD18-mediated adhesion is possible, this is followed by the
production of reactive oxygen species through the respiratory burst
[21
]. As shown in the past, reactive oxygen species are
able to induce apoptosis in neutrophils [22
,
23
]. In contrast, treatment with GM-CSF leads to
activation of CD11b/CD18 (Fig. 3A and 3B)
and induces adhesion, but is
by itself not a strong enough stimulus to incite a respiratory burst
[24
]. Stimulation with IFN- does not result in
activation or up-regulation of CD11b/CD18 (Fig. 3A
and 3B)
or in the
production of reactive oxygen species. Thus, whereas the activation of
the adhesion molecule CD11b/CD18 is necessary for the pro-apoptotic
effects of TNF-, it is not sufficient.
Moreover, GM-CSF was, like IFN-, not able to protect neutrophils
from the oxidative stress-induced apoptosis of high-dose TNF- (Fig. 5A and 5B)
. It is interesting that GM-CSF even enhanced pro-apoptotic
effects of TNF- (10–100 ng/mL). Thus, this cytokine, which by
itself is anti-apoptotic, promotes apoptosis when combined with
TNF-. Most likely this effect was to due to the ability of GM-CSF to
prime the neutrophils for enhanced generation of reactive oxygen
species induced by TNF-, because this effect was absent in
neutrophils from CGD patients (not shown).
The fact that the effect of TNF- was dose-dependent, and that CD11b
ligand binding can induce apoptosis, might account for the remarkable
differences that have been reported about the effects of TNF- on
neutrophil apoptosis [10
, 11
]. However, a
comparison of the doses of TNF- used in these and other studies did
not show a clear correlation between dose and effect on neutrophil
apoptosis. Most likely, other factors played a role as well, such as
differences in the purity or concentration of the TNF- preparations
used. Also, the purification of neutrophils can easily cause CD11b
activation [25
], leading to an increased tendency to
undergo a TNF--induced respiratory burst.
In contrast to earlier reports, baseline apoptosis of neutrophils on
which CD11b was absent or blocked [26
] and of cells from
CGD patients [22
] was similar to normal controls in our
hands. Significantly higher survival became apparent only after the
cells had been stimulated with a high dose of TNF-. Thus, in
contrast to what has been claimed before, neither the integrin CD11b
nor toxic oxygen metabolites seem to play a role in the spontaneous
apoptosis of neutrophils in vitro.
In general, it seems that pro-inflammatory cytokines, which are
normally present at inflammatory sites, protect neutrophils from
apoptosis [8
, 9
]. However, once a
respiratory burst has taken place, a more powerful signal may drive the
cells into apoptosis [22
, 23
]. In the case
of TNF-, adhesive interactions are essential for the induction of
the respiratory burst in neutrophils [21
]. Our findings
suggest that the interplay of stimulation by cytokines and adhesion
ultimately decides cell fate, as indicated by the combination of GM-CSF
and IFN- with different concentrations of TNF-.
The effect on the pathophysiology of reduced apoptosis after TNF-
stimulation of neutrophils in patients with LAD-1 and CGD has not been
studied. In LAD-1, where neutrophils do not reach the inflammatory site
at all, neutrophil apoptosis does not seem relevant. In CGD, one can
hypothesize that increased survival of activated neutrophils could play
a role in the formation of the granulomas characteristic for this
disease.
The reason that the respiratory burst provides such a strong impetus toward apoptosis could be that the leakage of reactive oxygen species is potentially self-damaging. It has been described that apoptosis of neutrophils caused by large amounts of toxic oxygen metabolites does not involve caspase activation [23 ], indicating that this apoptotic pathway may be specific for the neutrophil. It remains to be defined which cellular structures in the neutrophil are irreversibly damaged and lead to apoptosis.
In conclusion, the dual, concentration-dependent effects of TNF-
provide an explanation for previous controversial reports and support a
dominant role for TNF- in neutrophil apoptosis. The essential
process that shifts the balance toward apoptosis seems to be the
occurrence of a respiratory burst, which overrules other anti-apoptotic
stimuli.
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ACKNOWLEDGEMENTS |
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Received April 22, 2000; revised October 17, 2000; accepted October 19, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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