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Prepublished online as a Blood First Edition Paper on September 12, 2002; DOI 10.1182/blood-2002-01-0239.
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Blood, 15 January 2003, Vol. 101, No. 2, pp. 739-746
PHAGOCYTES
A role for apoptosis in the control of neutrophil homeostasis in
the circulation: insights from CD18-deficient mice
Pamela Weinmann,
Karin Scharffetter-Kochanek,
S. Bradley Forlow,
Thorsten Peters, and
Barbara Walzog
From the Department of Physiology, Freie
Universität Berlin, Berlin, Germany; Department of
Dermatology and Allergology, Universität Ulm, Ulm,
Germany; Department of Biomedical Engineering, University
of Virginia School of Medicine, Charlottesville; Department of
Dermatology, Universität zu Köln, Köln,
Germany; and Department of Physiology,
Ludwig-Maximilians-Universität, München,
Germany.
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Abstract |
The control of neutrophil turnover in the circulation is a key
event in homeostasis and inflammation. Using CD18- deficient (CD18 /) mice that show a
19-fold increase of blood neutrophil counts when compared with
wild-type animals
(CD18+/+), we found
that apoptosis of peripheral neutrophils was significantly reduced from
27.4% in the wild-type to 4.8% in
CD18/ mice within 4 hours
after isolation as measured by analysis of DNA content. This was
confirmed by detecting CD16 expression, nuclear morphology, and
internucleosomal DNA degradation. In contrast, no difference in
apoptosis was observed in neutrophils derived from the bone marrow.
Neutrophilia and delayed neutrophil apoptosis were also present in
CD18//interleukin 6 (IL-6/) double knockout
mice. Moreover, plasma of
CD18/ mice was not able
to delay apoptosis of CD18+/+
neutrophils and plasma of
CD18+/+ mice did not augment
apoptosis of CD18/
neutrophils. However,
CD18/ neutrophils
revealed an up-regulation of the antiapoptotic gene bcl-Xl and a down-regulation of the proapoptotic
gene bax- compared with
CD18+/+ neutrophils
suggesting that this delayed apoptosis. Accordingly, down-regulation of
Bax- using antisense technique delayed apoptosis and prolonged neutrophil survival. The replacement of the hematopoietic system of CD18+/+ mice by a
1:1 mixture of CD18+/+ and
CD18/ hematopoietic cells
abolished the delay of apoptosis in peripheral CD18/ neutrophils and
prevented neutrophilia. Altogether, this suggests that a delay of
neutrophil apoptosis in
CD18/ mice causes an
alteration of neutrophil homeostasis, which may induce the massive
increase of peripheral neutrophil counts. Thus, apoptosis seems to be
critically involved in the control of neutrophil turnover in the circulation.
(Blood. 2003;101:739-746)
© 2003 by The American Society of Hematology.
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Introduction |
Apoptosis (programmed cell death) represents the
key mechanism for the controlled elimination of cells within the body
and thus contributes to the maintenance of tissue homeostasis by
helping to keep the balance between cell proliferation and cell death. Apoptosis is an active and well-regulated process that is characterized by specific phenomena such as cell shrinkage, chromatin condensation, internucleosomal DNA fragmentation, membrane blebbing, and finally the
decay into apoptotic bodies.1,2 Human polymorphonuclear neutrophils (PMNs) die spontaneously by apoptosis within hours to days
but their lifetime can be shortened or extended by modulating apoptosis.3 Several inflammatory cytokines are known to
affect PMN apoptosis in vitro. The proinflammatory mediator tumor
necrosis factor (TNF-), for example, is known to reduce the life
span of human PMNs by accelerating apoptosis.4 Other
cytokines such as granulocyte-macrophage colony-stimulating factor
(GM-CSF) and granulocyte colony-stimulating factor (G-CSF) promote PMN
survival by inhibiting apoptosis.5 The intracellular
mechanism that controls the apoptotic machinery in PMNs subsequent to
cytokine stimulation involves the Bcl-2 family of apoptosis-associated genes, which consists of antiapoptotic (Bcl-2, Bcl-Xl,
Mcl-1, A1, etc) and proapoptotic members (Bax-, Bak, Bad,
Bcl-Xs, Bik, etc).6 The shift of the
balance between the proapoptotic factor Bax- and the antiapoptotic
factor Bcl-Xl toward the proapoptotic gene product is known
to promote apoptosis of human PMNs.4,7
In the situation in vivo, apoptosis of PMNs in the tissue is thought to
be critical for the final resolution of acute inflammation because it
prevents the uncontrolled release of their proinflammatory contents and
allows the nonphlogistic elimination of PMNs by macrophages and other
tissue cells.8-10 However, in addition to PMN infiltration of the tissue, the acute inflammatory response is accompanied by an
expansion of the peripheral PMN pool that is generally believed to be
due to an enhanced PMN production and maturation in the bone marrow an
effect that is mediated by cytokines such as G-CSF and
GM-CSF.11 However, several reports suggest that
inflammation-mediated neutrophilia is not simply due to enhanced
hematopoiesis. In patients with severe burns, circulating PMNs showed
impaired apoptosis that was induced by plasma factors up-regulating
GM-CSF levels.12 Cytokine-mediated delay of apoptosis in
peripheral PMNs was also observed in other inflammatory diseases
associated with neutrophilia such as cystic fibrosis and
pneumonia.7 Moreover, neutropenia in myelokathexis, a
congenital disorder, has been reported to involve a modulation, that
is, an enhancement of PMN apoptosis, which was associated with a
down-regulation of the antiapoptotic gene product
Bcl-Xl.13 Thus, a body of evidence indicates
that the dysregulation of PMN apoptosis may contribute to the expansion or the reduction of the peripheral PMN pool under pathologic
conditions. Together, this supports the concept that the control of PMN
apoptosis is not only involved in the final resolution of inflammation
by terminating the lifetime of the emigrated PMNs in the tissue and allowing their final elimination, but also seems to be critical for the
modulation of the peripheral PMN countat least under pathophysiologic
conditions. At least under pathophysiologic conditions, apoptosis seems
to be critical for the modulation of the peripheral PMN count. However,
little is known about the mechanisms that contribute to the maintenance
of PMN homeostasis under normal, noninflammatory conditions.
To address the question whether apoptosis may contribute to the
homeostasis of the functional PMN pool in the circulation under
noninflammatory conditions, we analyzed apoptosis of PMNs in an animal
model using CD18-deficient
(CD18/) mice and wild-type
(CD18+/+) mice with the same
genetic background as well as CD18/interleukin 6 double knockout
(CD18//IL-6/)
mice. In addition, PMN apoptosis was studied in
CD18+/+/CD18/
chimeric mice in which the hematopoietic system was replaced by a 1:1
mixture of CD18+/+ and
CD18/ hematopoietic cells.
Apoptosis was detected by measurement of CD16 expression on the cell
surface by analysis of DNA content and nuclear morphology as well as by
detection of DNA degradation (DNA-ladder). The role of the bcl-2 family
of apoptosis-associated genes was investigated by semiquantitative
reverse transcription-polymerase chain reaction (RT-PCR) and by
studying PMN apoptosis in the presence of antisense oligonucleotides.
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Materials and methods |
Isolation of murine PMNs
Murine PMNs were isolated from mutant mice deficient in CD18 or
wild-type control animals of the same genetic background (mixed 129/Sv
and C57BL/6J). In addition, CD18/IL-6 double-knockout mice and
wild-type animals with the same genetic background were used (mixed
129/Sv and C57BL/6J). All mice have been genotyped by Southern blot
analysis as described previously14 and were maintained under specific pathogen-free conditions in a barrier facility. Mice
were used at the age of 14 to 20 weeks. At this time point, the animals
were free of skin ulcerations and infections as measured by extended
microbiologic screening. Animal experiments were institutionally approved. Peripheral blood was collected by resection of the tip of the
tail, and aliquots of heparinized whole blood samples (200-700 µL)
were incubated with the fluorescein isothiocyanate (FITC)-conjugated anti-Gr-1 monoclonal antibody (mAb; final concentration 10 µg/mL) for 30 minutes at 4°C. Blood was incubated for 1 minute with 2 mL
lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM EDTA [ethylenediaminetetraacetic acid) at room temperature
and washed twice with 10 mL phosphate-buffered saline (PBS)-buffer
(PBS supplemented with 2 mM EDTA and 0.5% bovine serum albumin
[BSA]). Subsequently, samples (107 leukocytes) were
incubated for 20 minutes at 10°C with 10 µL magnetic beads coupled
to a monoclonal mouse anti-FITC antibody (anti-FITC microbeads,
Miltenyi Biotec, Bergisch Gladbach, Germany) and 90 µL PBS-buffer.
Cell separation was performed according to the supplier's protocol.
Briefly, after washing with PBS-buffer, cells were suspended in 500 µL PBS-buffer and loaded onto MACS MS separation columns (Miltenyi
Biotec) for magnetic separation. The column was washed 3 times with 1 mL PBS-buffer and was removed from the separator. Samples were eluted
twice by addition of 5 mL PBS-buffer. For isolation of PMNs from the
bone marrow, animals were killed by CO2 inhalation and the
bone marrow was harvested from tibias and femurs. Isolation of PMNs
from the bone marrow was performed as described for PMN isolation from
peripheral blood. Purity was more than 95% as measured by analysis of
Gr-1+ cells using flow cytometry. Isolated PMNs were also
analyzed for expression of CD18 to confirm the genotype (data not
shown). After the isolation procedure, PMNs (5 × 106/mL)
were cultured at 37°C in RPMI medium with HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer
supplemented with 10% fetal calf serum (FCS) unless indicated otherwise.
Separation of CD18+/+ and
CD18/ PMNs from
CD18+/+/CD18/
chimeric mice
For separation of CD18+ and
CD18 PMNs from the peripheral blood of
CD18+/+/CD18/
chimeric mice, cells were treated with the FITC-conjugated anti-Gr-1 mAb as described (see "Isolation of murine PMNs") and PMNs were isolated using the anti-FITC Multisort kit (Miltenyi Biotec). After
isolation of PMNs, beads were released from the PMNs according to the
supplier's instructions and PMNs were incubated for 15 minutes at
10°C with the phycoerythrin (PE)-labeled rat antimouse CD18 antibody
C71/16 and CD18+/+, and
CD18/ PMNs were separated
using magnetic beads coupled to a monoclonal mouse anti-PE antibody
(anti-PE microbeads, Miltenyi Biotec) as described.
Generation of
CD18+/+/CD18/
chimeric mice
Bone marrow cells were harvested from
CD18/ and wild-type mice
and transplanted into lethally irradiated wild-type mice as described
previously.15 Recipient mice (8 weeks old) were lethally irradiated in 2 doses of 600 rads each approximately 4 hours apart. Bone marrow cells from donor mice were harvested by flushing both femurs and tibias with RPMI without phenol red (Gibco, Grand Island, NY) containing 10% FCS (Atlanta Biologicals, Norcross, GA) under sterile conditions. Suspended bone marrow cells were washed and erythrocytes were lysed in 0.15 M NH4Cl lysing solution.
Approximately 4 million unfractionated bone marrow cells in 200 µL
media were delivered intravenously through the tail vein of each
recipient mouse. Recipient mice received transplants with a 1:1 mixture of CD18/ and wild-type
unfractionated bone marrow cells. Recipient mice were housed in a
barrier facility (individually ventilated cages, high-efficiency
particulate air [HEPA]) under pathogen-free conditions before
and after bone marrow transplantation. After bone marrow transplantation, mice were maintained on autoclaved water with antibiotics (5 mM sulfamethoxazole, 0.86 mM trimethoprim; Sigma Chemical, St Louis, MO) and fed autoclaved food.
Analysis of DNA content
DNA content was analyzed by flow cytometry (FACScan, Becton
Dickinson, Heidelberg, Germany) using propidium iodide (PI).
Briefly, isolated PMNs (5 × 105/100 µL) were washed
with PBS supplemented with 1 mM EDTA and resuspended in 70% ethanol.
PMNs were permeabilized overnight at  20°C, washed with PBS
supplemented with 1 mM EDTA, and suspended in 250 µL of the same
buffer. After addition of 20 µg/mL RNAase and 50 µg/mL PI (final
concentrations), samples were incubated for 15 minutes at room
temperature and kept at 4°C until flow cytometric analysis. In each
sample, 104 cells were counted and analyzed using Cell
Quest software (Becton Dickinson).
Cell surface expression of CD16
For analysis of CD16 expression of PMNs derived from
CD18+/+ or
CD18/ mice, aliquots of
whole blood (10-40 µL) were incubated for 1 hour at 4°C in the dark
with the PE-conjugated anti-CD16 mAb (final concentration of 10 µg/mL). Subsequently, samples were treated for 10 minutes in the dark
with 500 µL fluorescence-activated cell sorting (FACS) lysing
solution (Becton Dickinson), washed twice with PBS supplemented with 1 mM EDTA, and subjected to flow cytometry. In each sample,
104 cells were counted, gated off-line for granulocytes,
and analyzed using Cell Quest software. For analysis of CD16 expression
of PMNs derived from
CD18+/+/CD18/
chimeric mice, aliquots of whole blood (80-100 µL) were stained with
the PE-conjugated anti-CD18 mAb (final concentration of 10 µg/mL) and
the FITC-labeled anti-CD16 mAb (final concentration of 10 µg/mL) and
treated as described.
Analysis of nuclear morphology
Isolated PMNs were treated with acridine orange in a final
concentration of 5 µg/mL for 5 minutes at room temperature and investigated on a Nikon fluorescence microscope using an
epifluorescence adapter (Nikon DM510, B-2A; Duesseldorf, Germany) and
an 40/0.6 objective.
Internucleosomal DNA fragmentation assay
Isolated PMNs (107) from several mice (3-7) were
pooled after culture and lysed for 10 minutes on ice in 600 µL
hypotonic lysis buffer (10 mM EDTA, 0.2% Triton X-100, 10 mM Tris
[tris(hydroxymethyl)aminomethane], pH 7.5). After
centrifugation for 10 minutes at 4°C at 13 000g, DNA was
isolated by phenol/chloroform extraction and subsequent precipitation
with 2.5 volumes ethanol containing 0.1 M NaCl overnight at 20°C.
After centrifugation at 13 000g, the pellets were washed in
1 mL 70% ethanol, dried, and suspended in 20 µL H20.
After treatment with DNase-free RNase in a final concentration of 0.8 mg/mL for 30 minutes at 37°C, samples were analyzed by gel
electrophoresis in 1.8% agarose and visualized by ethidium bromide
staining under UV light.
RT-PCR
For isolation of RNA, about 600 µL blood was collected from
CD18+/+ mice that yielded
about 1 million PMNs after isolation using the magnetic bead separation
technique. About 20 million PMNs were obtained from an equal volume of
blood from CD18/ mice.
Total RNA was isolated by the guanidine isothiocyanate method16 using Trizol (Life Technologies, Eggenstein,
Germany), which yielded about 250 ng total RNA per 1 million PMNs. An
aliquot of 50 ng RNA was transcribed into cDNA using oligo(dT) primers (Amersham Pharmacia Biotech, Freiburg, Germany) and 50 U reverse transcriptase Moloney murine leukemia virus (MMLV; Promega,
Madison, WI). PCR amplification was carried out using specific primer
sets (TIB MOLBIOL, Berlin, Germany) for bcl-X (upstream primer:
5'-TTG-GAC-AAT-GGA-CTG-GTT-G; downstream primer:
5'-GTC-TGG-TCA-CTT-CCG-ACT-GA, 746-bp product); bax- (upstream
primer: 5'-CTG-AGC-AGA-TCA-TGA-AGA-CAG-G; downstream primer:
5'-CAG-TTG-AAG-TTG-CCG-TCA-G, 274-bp product), A1 (upstream primer:
5'-GAT-GGC-TGA-GTC-TGA-GCT-CA; downstream primer:
5'-GGC-AAT-CTG-CTC-TTG-TGG-AA, 330-bp product), bad (upstream primer:
5'-ATG-TTC-CAG-ATC-CCA-GAG-TT; downstream primer:
5'-TCA-CTG-GGA-GGG-GGC-GGA-GC, 490-bp product), and bak (upstream
primer: 5'-TGA-AAA-ATG-GCT-TCG-GGG-CAA-GGC; downstream primer:
5'-GTG-AAG-AGT-TCG-TAG-GCA-TT, 332-bp product). For control, a specific
primer set for  -actin (upstream primer: 5'-ATG-GCC-ACT-GCC-GCA-TCC-TC; downstream primer:
5'-CTA-GAA-GCA-CTT-GCG-GTG-CA, 430-bp product) was used. PCR
(30 [-actin] or 35 [others] cycles: 55 seconds 94°C, 55 seconds 60°C, 55 seconds 72°C) was performed using 1.25 U Ampli
Taq DNA polymerase (Perkin Elmer, Weiterstadt, Germany). PCR
products were analyzed by agarose gel electrophoresis and visualized
with ethidium bromide under UV light.
Antisense experiments
Isolated PMNs (5 × 106/mL) were incubated with a
mixture of 2 different bax antisense or scrambled oligonucleotides as
described previously for down-regulation of human bax.7
The sequence was adapted according to the murine gene sequence, and
oligonucleotides with a phosphorothioate backbone were used. (antisense
1: 5'-TCG-ATC-CTG-GAT-GAA-ACC-CT; antisense 2:
5'-TCC-CCA-GCC-ATC-CTC-CCT-GC; scrambled 1:
5'-TCA-GTC-CTG-GTA-GAA-CAC-CT; scrambled 2:
5'-CTC-ACC-CCA-CTT-CGC-CTC-GC). PMNs were treated with the
oligonucleotides in a final concentration of 20 µM each at 37°C in
RPMI medium without addition of FCS for the first 60 minutes of culture
to increase uptake of the oligonucleotides.
Detection of Bax- expression
After treatment with antisense or scrambled oligonucleotides as
described for antisense experiments, PMNs (5 × 105) were
permeabilized overnight at 20°C in 70% ethanol. After washing with
PBS supplemented with 1 mM EDTA, PMNs were suspended in 20 µL PBS supplemented with 1 mM EDTA and were incubated with the
polyclonal anti-Bax antibody in a final concentration of 10 µg/mL for
20 minutes at 4°C. After washing twice, samples were incubated with a
FITC-conjugated goat antirabbit IgG for 20 minutes at 4°C. After 2 washes, samples were subjected to flow cytometry (FACScan, Becton
Dickinson) and 104 cells were counted and analyzed using
Cell Quest software.
Antibodies
The PE-labeled rat antimouse CD18 antibody (clone C71/16), the
FITC-labeled rat antimouse Gr-1 antibody (clone RB6-8C5), the PE- and
the FITC-conjugated rat antimouse CD16 antibody (clone 2.4G2), and the
PE-conjugated rat antimouse CD14 antibody (clone rmC5-3) were obtained
from Pharmingen (San Diego, CA). The polyclonal anti-Bax antibody
(N-20) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The
FITC-conjugated goat antirabbit IgG was purchased form Sigma
(Deisenhofen, Germany). The polyclonal goat antimouse G-CSF antibody
and the control goat IgG were obtained from R & D Systems
(Wiesbaden-Nordenstadt, Germany).
Reagents
Acridine orange (3,6-Bis-(dimethyl-amino)-acridine), BSA,
DNase-free RNase, ethidium bromide, penicillin, PI, RNase A,
streptomycin, and Triton X-100 were obtained from Sigma (Deisenhofen,
Germany). Buffers, cell culture media, and FCS were obtained from
Biochrom (Berlin, Germany). The gyrase inhibitor Baytril containing 25 mg/mL enrofloxacin was obtained from Bayer (Leverkusen, Germany). The
bax- antisense and scrambled oligonucleotides were obtained from TIB
MOLBIOL. The enzyme-linked immunosorbent assay (ELISA) kits
(KMC2011 for mouse GM-CSF and KMC4021 for mouse interferon- [IFN-]) were purchased from Biosource (Ratingen, Germany).
Statistical analysis
Data shown represent mean ± SD where applicable.
Statistical significance was determined using the Student
t test.
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Results |
Alteration of blood PMN homeostasis in
CD18/ mice
The analysis of leukocyte counts in the peripheral blood of
CD18/ mice and wild-type
control animals (Figure 1A) showed that
total leukocyte counts were about 9-fold elevated in the deficient
animals (84.2 × 103/µL) compared with wild-type
controls (9.6 × 103/µL), a result that is in
accordance with previous observations.14,17 This effect
was largely due to neutrophilia with about a 19-fold increase of
granulocytes from 3.1 × 103/µL in wild-type controls
to 58.0 × 103/µL in
CD18/ mice (Figure 1B).
However, lymphocyte counts (3.5-fold) as well as monocyte counts
(11-fold) were elevated in the gene-deficient animals compared with
controls. Although extended microbiologic screening had shown that the
animals used had no infections (data not shown), we studied the effect
of the antibiotic enrofloxacin on the leukocyte counts. A final
concentration of 0.01% enrofloxacin in the drinking water over 13 days
slightly diminished the PMN counts in the circulation of the mutant
mice (n = 4) as measured at day 2, 5, 7, and 13 of enrofloxacin
treatment (data not shown). However, the effect was not significant and
PMN counts remained several-fold elevated when compared with
enrofloxacin-treated control animals (n = 8). Moreover, PMN counts in
the wild-type animals were slightly reduced in the presence of
enrofloxacin. Together, this may further confirm that the increase of
leukocyte counts in the absence of CD18 was not due to the activation
of host defense mechanisms caused by bacterial infections.
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| Figure 1.
Neutrophilia in
CD18/ mice.
Total (A) and differential leukocyte counts (B) in whole blood samples
obtained from CD18+/+
(n = 23) or CD18/ mice
(n = 21). Data represent means ± SDs;
**P < .001.
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Delayed apoptosis of blood PMNs in
CD18/ mice
To prove the hypothesis that a delay of apoptosis may contribute
to the massive accumulation of neutrophils in the circulation, we
measured the loss of DNA content, a well known marker of
apoptosis,1 in PMNs that were isolated from the
circulation of CD18/ or
CD18+/+ animals. Figure
2A shows the data obtained in one
representative experiment. Within 4 hours after the onset of culture,
the original fluorescence histogram revealed a decrease of the gDNA
content in 27.8% of PMNs derived from the circulation of wild-type
mice, whereas only 3.4% of mutant PMNs showed a loss of DNA content within the same time period. Figure 2B shows the mean values of the
observed effect in PMNs from 7 CD18+/+ mice and 5 CD18/ mice. The
quantitative analysis revealed that 0.8% of PMNs freshly isolated from
the circulation of wild-type animals (0 hours) showed a loss of DNA
content. In contrast, only 0.2% of the PMNs derived from the
circulation of mutant mice were apoptotic. Within 4 hours after the
onset of culture of the freshly isolated PMN, 27.4% of wild-type cells
underwent apoptosis, whereas only 4.8% of the PMNs isolated from the
circulation of CD18/ mice
showed a loss of DNA content. This corresponds to a reduction of
apoptosis in absence of CD18 to 17.5% compared with wild-type controls
(100%). To confirm this observation, we measured the down-regulation
of CD16 (Fc receptor type III) expression on the cell surface as a
marker for PMN apoptosis in whole blood samples.18 Figure
3A shows one representative fluorescence
histogram of the results obtained. When aged in culture for 8 hours,
30.1% of PMNs obtained from the circulation of wild-type animals
showed a decrease of CD16 expression on the cell surface. In contrast, only 6.8% of PMNs from mutant mice had a substantially reduced CD16
expression. Figure 3B shows the mean values obtained from 13 wild-type
animals and 11 CD18-deficient mice. The data revealed that 5.4% of
PMNs freshly isolated from the circulation (0 hours) of wild-type
animals showed low CD16 expression on the cell surface. In contrast,
only 0.6% of PMNs from the mutant mice revealed diminished CD16
expression. Similar results were obtained at later time points (4, 8, and 22 hours after the onset of culture) with 13.1%, 32.3%, and
65.7% of PMNs with low CD16 expression in the wild-type and 1.4%,
8.7%, and 42.4% of PMNs showing reduced CD16 expression in the
absence of CD18. Thus, the delay of neutrophil apoptosis was not only
detectable in isolated PMNs but also in PMNs of whole blood samples,
demonstrating that the observed effect was not due to the
isolation procedure.
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| Figure 2.
A delay of apoptosis in blood PMNs of
CD18/ mice.
Flow cytometric analysis of DNA content measured in propidium
iodide-stained PMNs isolated from the circulation of
CD18+/+ (n = 7) or
CD18/ mice (n = 5). (A)
Representative recording of the fluorescence histograms obtained from
isolated blood PMNs aged for 4 hours in culture without further
stimulation. Numbers indicate apoptotic PMNs in percent of total cell
number. (B) Mean values obtained after 0 hours and 4 hours of culture.
Data represent means ± SDs; **P < .005.
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| Figure 3.
Delayed apoptosis of PMNs in
CD18/ mice is also
apparent in whole blood samples.
Flow cytometric analysis of CD16 expression on the cell surface of PMNs
using the PE-labeled anti-CD16 mAb measured in whole blood samples
obtained from CD18+/+
(n = 13) or CD18/ mice
(n = 11). (A) Representative recording of the fluorescence histograms
obtained from PMNs aged for 8 hours in culture without further
stimulation. Numbers indicate apoptotic PMNs with low CD16 expression
in percent of total cell number. (B) Mean values obtained after 0, 4, 8, or 22 hours of culture. Data represent means ± SDs;
**P < .005.
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To confirm the delay of PMN apoptosis in
CD18/ mice, we studied the
morphology of the nuclei by fluorescence microscopy after acridine
orange staining (Figure 4A). Almost all
PMNs isolated from the circulation of wild-type animals showed highly
condensed nuclei (arrows) within 4 hours after the onset of culture,
which is typical for apoptotic PMNs. In contrast, the majority of PMNs from CD18/ mice showed
segmented nuclei within this time period, which is typical for mature,
nonapoptotic PMNs and only some PMNs showed condensation of the nuclei.
Next, the degradation of gDNA was studied (Figure 4B). And again, only
PMNs derived from wild-type animals showed strong DNA laddering within
4 hours after the onset of culture, whereas this effect was barely
detectable in PMNs derived from
CD18/ mice. Thus, we were
able to demonstrate a substantial delay of PMN apoptosis in the
peripheral blood of CD18/
mice compared with PMNs from wild-type control animals by the use of 4 independent methods.
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| Figure 4.
Analysis of nuclear morphology and DNA degradation.
(A) Epifluorescence photomicrographs of isolated blood PMNs from
CD18+/+ mice or
CD18/ mice stained with
acridine orange 4 hours after the onset of culture. Results are
representative of 3 independent experiments. (B) Agarose gel of
low-molecular-weight DNA of isolated blood PMNs from
CD18+/+ or
CD18/ mice 4 hours after
the onset of culture. Results are representative of 3 independent
experiments.
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To find out whether the observed delay of apoptosis in the peripheral
PMNs was due to an effect that was already induced in the bone marrow
prior to the release of the PMNs into the circulation, apoptosis was
studied in PMNs isolated from the bone marrow. Using PMNs derived from
6 CD18-deficient animals and 5 wild-type mice, no significant
difference in apoptosis was detectable by measuring the DNA content of
isolated PMNs using propidium iodide (Figure 5). This was true for freshly isolated
PMN (0 hours) as well as for PMNs that were cultured for 4, 8, and 22 hours, respectively. Thus, the delay of apoptosis in the
blood PMNs of the mutant mice was not a consequence of an effect in the
bone marrow, demonstrating that the absence of CD18 has an impact on
apoptosis not before PMNs are released into the circulation.
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| Figure 5.
No difference of apoptosis in PMNs derived from the bone
marrow.
Apoptosis as measured by detecting the loss of DNA content by flow
cytometry using PI-stained PMNs isolated from the bone marrow of
CD18+/+ (n = 5) or
CD18/ mice (n = 6) after
0, 4, 8, or 22 hours of culture. Data represent means ± SDs;
NS indicates not significant.
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Because GM-CSF is postulated to cause neutrophilia in man by
inhibiting apoptosis of PMNs,7 we studied the
concentration of GM-CSF in the plasma of
CD18+/+ and
CD18/ mice by means of the
ELISA technique (Figure 6). The analysis of the GM-CSF concentration showed that there was no significant difference in the concentration of GM-CSF in the plasma of both animal
groups. Thus, an increase of the plasma levels of GM-CSF seems not to
be involved in the delay of PMN apoptosis in the mutant mice. We also
measured the concentration of IFN- in the plasma of
CD18+/+ and
CD18/ mice using an ELISA
technique and found that it was below the detection limit of 1 pg/mL in
both groups, suggesting that an increased IFN- level is not involved
in the observed delay of apoptosis (data not shown). However, IL-6 is
thought to promote PMN survival by inhibiting apoptosis,19
and the plasma concentration of IL-6 has been previously reported to be
elevated in CD18/ mice
when compared with wild-type control animals.14 Therefore, we analyzed the leukocyte counts in the peripheral blood of
double-knockout mice that were deficient in both CD18 and IL-6
(CD18//IL-6/
mice). Similar to
CD18/ mice, the
CD18//IL-6/
mice showed leukocytosis (Table 1). In
contrast to CD18/ mice,
which revealed a massive neutrophilia as well as increased monocyte and
lymphocyte counts, the
CD18//IL-6/
mice showed only a neutrophilia but no increase of monocyte or lymphocyte counts when compared with the wild-type control animals. However, because neutrophilia was also present in
CD18//IL-6/
mice, it seems rather unlikely that IL-6 alone is responsible for the
observed effect in CD18/
mice. Moreover, PMNs obtained from the circulation of
CD18// IL-6/
mice showed also a significant delay of apoptosis when compared with
PMNs from wild-type animals as measured by detecting the loss of CD16
expression on the cell surface as well as the DNA content
(Table 2). Together, this shows
that IL-6 is at least not required to induce the neutrophilia or the
delay of PMN apoptosis in the animal models used.
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| Figure 6.
Plasma concentrations of GM-CSF.
Plasma concentration of GM-CSF as measured by ELISA technique in
CD18+/+ (n = 20) or
CD18/ mice (n = 14).
Data represent means ± SDs; NS indicates not significant.
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However, other plasma factors besides GM-CSF, IFN-, and IL-6 are
known to cause a delay of PMN apoptosis.7,19 Therefore, we
studied whether the plasma at all may have an effect on PMN apoptosis.
PMNs freshly isolated from the circulation of
CD18+/+ mice or
CD18/ mice were incubated
in the presence of plasma derived from
CD18+/+ or
CD18/ mice, respectively
(Figure 7). It turned out that treatment
of blood PMNs with plasma from both groups had absolutely no effect. Neither plasma derived from mutant mice was able to reduce apoptosis of
wild-type PMNs when compared with apoptosis of wild-type PMNs treated
with wild-type plasma nor was plasma from wild-type mice able to
enhance apoptosis of mutant PMNs when compared with the effect of
plasma from mutant mice. This suggests that soluble factors in the
plasma were not sufficient to impair apoptosis of PMNs in
CD18-deficient animals. However, we performed additional experiments in
which G-CSF in plasma of
CD18/ mice was neutralized
using a goat antimouse G-CSF antibody. When compared with plasma alone
or plasma in the presence of a goat control IgG, neutralizing G-CSF in
plasma of CD18/ mice had
no effect on apoptosis of PMNs isolated from the peripheral blood of
wild-type mice (data not shown). However, to further exclude the
possibility that mutant mice have a soluble factor in the plasma that
triggers a delay of apoptosis in mutant PMNs in the bone marrow, which
might not have a detectable effect until the PMNs reach the
circulation, we analyzed the effect of plasma from mutant mice on PMNs
from the bone marrow of wild-type mice. However, this had no detectable
effect on PMN apoptosis. Because bone marrow PMNs from wild-type
animals did not respond to plasma from mutant mice with respect to a
delay of apoptosis, this further supports the view that soluble factors
in the plasma are probably not sufficient to delay PMN apoptosis in the
absence of CD18.
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| Figure 7.
Soluble factors in the plasma had no effect on PMN
apoptosis.
Flow cytometric analysis of DNA content measured in PI-stained PMNs
isolated from the circulation of
CD18+/+ mice (left),
CD18/ mice (middle), or
the bone marrow of CD18+/+
mice (right) in the presence of RPMI medium (without FCS) supplemented
with 50% plasma from CD18+/+
mice or CD18/
mice. Data represent apoptotic cells in percent of apoptosis
in the presence of wild-type plasma (100%) 8 hours after the
onset of culture. Data represent means ± SDs; NS indicates not
significant.
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Control of apoptosis by the bcl-2 family of
apoptosis-associated genes
Next, we investigated the molecular mechanism that underlies the
delay of PMN apoptosis in the circulation of CD18-deficient animals.
Because the bcl-2 family of apoptosis-associated genes is critically
involved in the control of apoptosis of human PMNs,4 we
studied whether a shift of balance between proapoptotic and antiapoptotic members of this gene family may be also responsible for
the delay of PMN apoptosis in vivo. Using semiquantitative RT-PCR, we
found an up-regulation of the antiapoptotic factor bcl-Xl
and a down-regulation of the proapoptotic gene product bax- in
peripheral PMNs of mutant mice when compared with PMNs obtained from
wild-type animals (Figure 8A). In
contrast, no striking difference in the expression of A1, bad, or bak
was observed between neutrophils from
CD18/ animals when
compared with control cells (data not shown) suggesting that the
differential expression of these factors is not critical for the
observed delay of apoptosis. However, to further characterize the shift
of balance between bcl-Xl and bax-, the
bcl-Xl/bax- ratio was calculated using the optical
density of the PCR products obtained (Figure 8B). The calculation
revealed a shift of the bcl-Xl/bax- ratio from 0.4 in
control PMNs to 3.1 in
CD18/ PMNs, which
corresponds to a 7.8-fold increase of the ratio compared with control.
This suggests that the profound shift of balance between these factors
toward the antiapoptotic gene product may be involved in the observed
delay of apoptosis in
CD18/ PMNs. To prove
whether this shift of balance may be sufficient to delay apoptosis, we
used the antisense technique using bax- antisense oligonucleotides
and scrambled oligonucleotides. As assessed by flow cytometry after
intracellular immunofluorescence staining of Bax- in permeabilized
PMNs, we found a substantial down-regulation of Bax- after antisense
treatment when compared with the effect of the scrambled
oligonucleotides (Figure 8C). This down-regulation of Bax-, which
mimicked the shift of balance observed in
CD18/ PMNs, was sufficient
to prolong the lifetime of PMNs (Figure 8D). The analysis revealed that
17.2% of the cells survived within 24 hours after antisense treatment.
In contrast, only 10.8% of the cells were nonapoptotic in the presence
of scrambled antisense oligonucleotides. Thus, the survival rate was
1.6-fold increased in the presence of the antisense oligonucleotides
demonstrating that a decrease of bax- expression promotes PMN
survival by delaying apoptosis. This shows that the observed shift of
the balance observed in PMNs from
CD18/ mice is sufficient
to impair PMN apoptosis.
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| Figure 8.
Decreased bax- expression caused the delay of PMN
apoptosis.
(A) Differential expression of bcl-Xl and bax- in 4 different PMN samples (1-4) freshly isolated from the circulation of
CD18+/+ mice or
CD18/, respectively, as
measured by semiquantitative RT-PCR. (B) Alteration of the
bcl-Xl/bax- ratio on apoptosis as calculated from the
mean optical density of the PCR products obtained from PMNs of
7 animals of each group. Numbers indicate the mean ratios ± SDs;
**P < .005. (C) Flow cytometric analysis of the
down-regulation of Bax- in the presence of antisense
oligonucleotides (bax--as) when compared with the effect of
scrambled oligonucleotides (bax--sc). (D) Survival of wild-type PMNs
in the presence of bax- antisense (bax--as) or scrambled
(bax--sc) oligonucleotides. Data represent means ± SDs;
**P < .005; n = 10.
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Finally, we addressed the question whether the observed delay of
apoptosis is a primary effect that is directly caused by the absence of
CD18 and measured PMN apoptosis in
CD18+/+/CD18/
chimeric mice. First of all, we analyzed the peripheral PMN counts of
these animals and found a mild leukocytosis in these animals (Figure
9A), which was due to the elevation of
the lymphocyte counts (Figure 9B). In contrast, no difference in the
peripheral PMN or monocyte counts was detectable when compared with
the wild type. To study apoptosis of PMNs derived from the
CD18+/+/CD18/
chimeric mice, PMNs were isolated from the peripheral blood of these animals and CD18/
and CD18+/+ PMNs were
separated. Subsequently, cells were subjected to the analysis of the
intracellular DNA content (Figure 10A).
No difference of apoptosis was detectable between the
CD18/ and the
CD18+/+ PMNs. A similar result
was obtained when PMN apoptosis was measured in whole blood samples by
detecting the diminished cell surface expression of CD16 (Figure 10B).
After double staining with a FITC-labeled anti-CD16 mAb and a
PE-labeled anti-CD18 mAb, no significant difference of apoptosis
between the CD18/ and
CD18+/+ PMNs in the peripheral
blood of
CD18+/+/CD18/
chimeric mice was detectable. Thus, the presence of wild-type leukocytes abolished the delay of apoptosis in
CD18/ PMNs of the chimeric
animals, suggesting that the observed delay of PMN apoptosis in
CD18/ mice is not simply a
cellular effect.
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| Figure 9.
Peripheral blood leukocyte counts in
CD18+/+/CD18/
chimeric mice.
Total (A) and differential leukocyte counts (B) in whole blood samples
obtained from
CD18+/+/CD18/
chimeric mice (n = 10) or wild-type control animals (n = 10). Data
represent means ± SDs; **P < .01; NS indicates not
significant.
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| Figure 10.
Apoptosis of
CD18+/+ and
CD18/ PMNs derived from
CD18+/+/CD18/
chimeric mice.
(A) Flow cytometric analysis of DNA content measured in PI-stained
CD18+/+ and
CD18/ PMNs isolated from
the circulation of
CD18+/+/CD18/
chimeric mice and aged for 4 hours in culture without further
stimulation (n = 3). Data represent apoptotic PMNs with a loss of DNA
content in percent of total cell number. Data represent means&nbs |
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Abstract
Introduction