This information is current as
of June 17, 2017.
Coronin-1 Is Associated with Neutrophil
Survival and Is Cleaved during Apoptosis:
Potential Implication in Neutrophils from
Cystic Fibrosis Patients
Sandra Moriceau, Chahrazade Kantari, Julie Mocek, Noélie
Davezac, Julie Gabillet, Ida Chiara Guerrera, Frank
Brouillard, Danielle Tondelier, Isabelle Sermet-Gaudelus,
Claire Danel, Gérard Lenoir, Soizic Daniel, Aleksander
Edelman and Véronique Witko-Sarsat
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2009 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2009; 182:7254-7263; ;
doi: 10.4049/jimmunol.0803312
http://www.jimmunol.org/content/182/11/7254
The Journal of Immunology
Coronin-1 Is Associated with Neutrophil Survival and Is
Cleaved during Apoptosis: Potential Implication in Neutrophils
from Cystic Fibrosis Patients1
Sandra Moriceau,2* Chahrazade Kantari,2* Julie Mocek,2* Noélie Davezac,† Julie Gabillet,*
Ida Chiara Guerrera,* Frank Brouillard,* Danielle Tondelier,* Isabelle Sermet-Gaudelus,‡
Claire Danel,§ Gérard Lenoir,‡ Soizic Daniel,* Aleksander Edelman,* and Véronique Witko-Sarsat3*
N
eutrophils are professional phagocytic cells that are able
to phagocytose and destroy infectious agents through
oxygen-dependent or -independent mechanisms (1).
They are key actors in anti-infectious host defense that can modulate the immune response (2) but are also inflammatory cells that
can mediate tissue damage (3, 4). Using gene microarray approaches, it has been shown that during phagocytosis of a pathogen, neutrophils can activate a transcriptional program that ultimately results in their apoptosis (5). Those data provided evidence
of a link between bactericidal mechanisms and apoptosis, thereby
suggesting a more complex regulation of neutrophil apoptosis than
previously anticipated. Thereafter, macrophages phagocytose apoptotic neutrophils to eliminate them from the site of inflammation. This latter step should be tightly regulated because it is a key
step in the resolution of inflammation (6). Indeed, neutrophil ap-
*Institut National de la Santé et de la Recherche Médicale Unité 845, Université René
Descartes, Hôpital Necker, Paris, France; †Centre National de la Recherche Scientifique FRE 2937 Institut André Lwoff, Villejuif, Université Paris-sud, Paris, France;
‡
Service de Pédiatrie Générale, Hôpital Necker-Enfants Malades, Paris, France; and
§
Service d’anatomopathologie, Hôpital Bichat, Paris, France
Received for publication October 2, 2008. Accepted for publication March 25, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
optosis appears to be a potential target for anti-inflammatory therapy and identification of proteins that control neutrophil survival
represent a promising avenue of research (7). For example, in the
neutrophil-dominated inflammation associated with cystic fibrosis
(CF),4 neutrophils persist in the patients airways and are an important factor in a patient’s prognosis (8). We and others (9, 10)
previously showed that circulating neutrophils from CF patients
are activated in the absence of detectable infection and that this
activation is potentiated by chronic pulmonary Pseudomonas
aeruginosa infection. Moreover, several lines of evidence suggest
that CF patient’s neutrophils undergo delayed apoptosis, thus potentiating the inflammatory state (11, 12).
Despite the cardinal importance of controlling the neutrophil
survival/apoptosis balance, the complex molecular pathways involved in neutrophil apoptosis are still poorly understood but involved some neutrophil-specific mechanisms (13). Recent findings
strongly suggest that neutrophils can succumb to several types of
programmed cell death which can be caspase dependent or independent (14, 15).
The aims of this study were to identify, by a proteomic approach, cytosolic proteins modified during neutrophil apoptosis
and to investigate whether modification of a given protein was
only a consequence or part of the apoptotic process, i.e., if the
modified protein could modulate apoptosis itself.
1
This work was supported by research funding from the European project NEUPROCF and the associations “Mucoviscidose ABCF,” “Vaincre La Mucoviscidose,”
and the Chancellerie des Universités (Legs Poix).
2
S.M., C.K., and J.M. contributed equally to the work.
3
Address correspondence and reprint requests to Dr. Véronique Witko-Sarsat, Institut
National de la Santé et de la Recherche Médicale Unité 845, Hôpital Necker, 149, rue
de Sèvres, 75015 Paris Cedex 15, France. E-mail address: [email protected]
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803312
4
Abbreviations used in this paper: CF, cystic fibrosis; 7-AAD, 7-aminoactinomycin
D; DIOC6, 3,3⬘dihexyloacarbocyanine iodide; DMF, dimethylformamide; HA, hemagglutinin A; PFA, paraformaldehyde; pI, isoelectric point; PVDF, polyvinylidene
fluoride.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Because neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival
should provide new strategies to modulate inflammation. Using a proteomic approach, coronin-1 was identified as a cytosolic
protein cleaved during neutrophil apoptosis. Coronin-1 is an actin-binding protein that can associate with phagosomes and
NADPH oxidase, but its involvement in apoptosis was currently unknown. In coronin-1-transfected PLB985 cells, coronin-1
overexpression did not modify the kinetics of granulocyte differentiation as assessed by CD11b labeling. Concerning apoptosis,
increased coronin-1 expression in dimethylformamide-differentiated PLB985 significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. Likewise, coronin-1 significantly decreased TRAIL-induced apoptosis with less
mitochondrial depolarization, caspase-3 and caspase-9 activities, but not caspase-8 or Bid truncation suggesting that coronin-1
interfered with mitochondria-related events. To validate the prosurvival role of coronin-1 in a pathophysiological condition
involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (CF) patients were studied. Circulating neutrophils
from CF patients had more coronin-1 expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. This was
associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and
mitochondria depolarization. In addition, inflammatory neutrophils from CF patients lungs showed an intense coronin-1 immunolabeling. We concluded that coronin-1 could constitute a potential target in resolving inflammation. The Journal of Immunology, 2009, 182: 7254 –7263.
The Journal of Immunology
7255
We herein focused on coronin-1, also known as p57 (16) or
TACO for tryptophan aspartate-containing coat protein (17),
which is an actin-binding protein exclusively expressed in leukocytes and very abundant in neutrophils, thereby suggesting a cell
type-specific function. Coronin-1 was discovered as a 57-kDa protein that copurified with phospholipase activity (16). In neutrophils, it was associated with actin and the p40phox subunit of the
NADPH oxidase complex (18). In macrophages, it associates with
phagosomes (17). Furthermore, loss of coronin-1 activity, either
using a dominant-negative construct or by knockdown using short
interfering RNAs inhibited the internalization, but not the binding,
of IgG-opsonized RBC by RAW 264.9 cells (19). These data are
consistent with a coronin-1 role in mediating actin dynamics in a
variety of processes in hemopoietic cells but its potential implication in apoptosis was currently unexplored.
Materials and Methods
Neutrophil isolation, PLB985 culture, and transfection
Apoptosis induction and quantification
To induce physiological apoptosis, neutrophils were resuspended at 2 ⫻
106/ml in RPMI 1640 (Invitrogen Life Technologies) supplemented with
10% FCS and incubated for 15 h (or the indicated times for kinetic studies)
at 37°C in a humidified atmosphere with 5% CO2. Phosphatidylserine externalization was assessed by flow cytometry after PE-conjugated annexin-V (PE-annexin V) and 7-aminoactinomycin D (7-AAD) labeling as
described previously (22). Apoptosis-induced phosphatidylserine externalization was quantified by PE-annexin-V (FL2) labeling after necrotic
7-AAD-labeled cells (FL3) were excluded from the analysis using a FACScan flow cytometer (CellQuest software and BD Immunocytometry Systems). Mitochondrial depolarization was evaluated by flow cytometry after
3,3⬘-dihexyloxacarbocyanine iodide (DIOC6) labeling (1 ␮M in RPMI
1640), according to the manufacturer’s instructions (Sigma-Aldrich).
When indicated, neutrophils were incubated either with the pancaspase
inhibitor Z-VAD-FMK (50 –200 ␮M; Bachem) or with the caspase-3 inhibitor Z-DEVDL-D-FMK (10 –200 ␮M; Bachem) or with the caspase-9
inhibitor Z-LEHD-FMK (10 –100 ␮M; Calbiochem). Neutrophils were
also incubated with the chemokine IL-8 (50 –100 ng/ml; PeproTech), GCSF (10 –50 ng/ml), or TNF-␣ (5 and 50 ng/ml).
PLB985 apoptosis was triggered by adding gliotoxin (2 ␮g/ml; SigmaAldrich), which induces Bak dimerization (23) and interferes with the
NF-␬B pathway (24) or by TRAIL (10 ng/ml; R&D Systems) that triggers
death receptor-dependent apoptosis (25), for the indicated times. Apoptosis-induced chromatin condensation was assessed by DNA staining with
the blue fluorescent dye Hoechst (5 ␮g/ml; Sigma-Aldrich). Cells harbor-
FIGURE 1. Modulation of cytosolic proteins (p38MAPK, gelsolin, and
moesin) during neutrophil apoptosis. A, Phosphatidylserine externalization
was detected with PE-conjugated annexin V, plotted vs cell necrosis, and
assessed by 7-AAD labeling. Under basal conditions, little externalization
(3%) was detected on neutrophils, whereas after apoptosis induction, significantly more annexin V-labeled neutrophils (71%) were observed. B,
Western blot analysis of caspase-3 activation. Identification of p38MAPK
(C), gelsolin (E), and moesin (G) as spots on 2D gels of neutrophil cytosolic proteins that were smaller after apoptosis than under basal conditions
and validation by Western blot analysis using respective specific Abs (D,
F, and H). The full-length protein is shown by an arrow, which indicates
its apparent molecular mass. When the fragments are observed on a Western blot (for gelsolin and moesin), their apparent molecular mass are indicated by another arrow. The figure reports representative results obtained
with neutrophils from a given subject that were confirmed in independent
experiments conducted with three different neutrophil preparations from
three different subjects.
ing nuclei with condensed chromatin were considered apoptotic (26).
Caspase-3, caspase-8, and caspase-9 activities were measured in PLB985
cells using colorimetric assays (Biovision) with their specific specific substrates i.e., DEVD-pNA, IETD-pNA and LEHD-pNA, respectively.
Isoelectric focusing, SDS-PAGE analysis, and mass
spectrometry
The cytosolic fraction was obtained after low-level sonication (47 Hz for
20 s at 4°C) of the neutrophil pellet resuspended at 100 ⫻ 106/ml in
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Human neutrophils were isolated from the EDTA-anticoagulated blood of
healthy volunteers (Etablissement Français du Sang, Paris, France) or CF
patients using density-gradient centrifugation on polymorphprep (Nycomed), as described previously (20). Twenty-seven CF patients, 11 girls
and 16 boys (mean age ⫾ SEM, 12.1 ⫾ 0.95 years), who are followed in
the General Pediatrics Department of Necker-Enfants Malades Hospital,
were recruited for the study. CF was diagnosed according to standard criteria, including a sweat test. Genotyping of CF patients showed the following allelic frequencies: 52% ␦F508 homozygote, 15% ␦F508 heterozygote with an unknown mutation, and 33% with other mutations in both
alleles. All CF patients had chronic Pseudomonas aeruginosa colonization.
Patients or their parents gave their written informed consent to participate
in the study. This study was approved by the Ethics Committee of NeckerEnfants Malades (Paris, France).
Cells of the promyelocytic cell line, henceforth called PLB985, were
grown in RPMI 1640 medium supplemented with 2 mM glutamine, 10%
FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin in humidified incubator at 37°C, 5% CO2 in air. PLB985 were transfected using Amaxa
technology according to the manufacturer’s instructions, with the plasmids
pcDNA3, pcDNA3coroHA-Nter, or pcDNA3coroHACter (GeneCopoeia)
to express, respectively, no recombinant protein (a negative control) or
coronin-1 expressing the hemagglutinin A (HA) tag at the amino-terminal
(CoroNter) or carboxyl-terminal (CoroCter). After 1 mo of culture in the
presence of neomycin (1 mg/ml), whose gene is encoded by the plasmid,
stably transfected PLB985 expressing either CoroNter or CoroCter were
selected. For granulocytic differentiation, wild-type or coronin-1-expressing PLB985 were exposed to 0.5% dimethylformamide (DMF) for 6 days
as described previously (21).
7256
ANTIAPOPTOTIC EFFECT OF CORONIN-1 IN NEUTROPHILS
Table I. Characteristics of the 11 proteins modified by apoptosis induction and cut from the
two-dimensional electrophoresis gel
Observed
Theoretical
Protein name
pI
kDa
pI
kDa
Scorea
Gelsolin humanb
Gelsolin humanb
Gelsolin humanb
Moesin humanb
Moesin humanb
MAPK p38 humanb
Coronin-1 humanb
Annexin A1 humanc
Pyruvate kinase humanc
Coronin-1 humanc
Annexin A1 humanc
5.5–6
5.7–6
5.7–6
6.5–7
6.5–7
5.5–6
6–6.5
5.8–6.2
5.5–6
5.8–6.2
5.3–5.7
75–100
75–100
75–100
60–80
60–80
35–45
40–55
35–45
30–40
23–30
25–35
5.9
5.9
5.9
6.09
6.09
5.76
6.25
6.64
9
6.25
6.64
85.7
85.7
85.7
67.7
67.7
40.2
51
38.6
57.8
51
38.6
171
228
198
142
141
36
115
105
138
92
144
Coverage
(%)
33.2
36.6
34.8
34
15
26
26.7
26
44.3
Swiss-Prot entry name
GELS_HUMAN
GELS_HUMAN
GELS_HUMAN
MOES_HUMAN
MOES_HUMAN
MAPK5_HUMAN
COR1A_HUMAN
ANXA1_HUMAN
KPYM_HUMAN
COR1A_HUMAN
ANXA1_HUMAN
a
These scores are indicated by MASCOT.
Spots smaller in apoptotic neutrophils.
c
Spots larger in apoptotic neutrophils.
b
Western blot analysis
Neutrophil or PLB985 cell lysates were subjected to Western blot analysis,
as described previously (29). The primary Abs were as follows: rabbit
polyclonal anti-p38MAPkinase (Cell Signaling Technology), mouse
monoclonal anti-gelsolin (Sigma-Aldrich), mouse monoclonal anti-moesin
(Sigma-Aldrich), mouse monoclonal anti-coronin-1 (Abnova), rabbit polyclonal anti-Bid (Santa Cruz Biotechnology), and rabbit polyclonal anti-HA
(Roche). The membrane was stripped in Restore Buffer solution (Pierce)
and reprobed with mouse monoclonal anti-␣-actin 2 Ab (Sigma-Aldrich) or
anti-GAPDH Abs (Abcys) (1 ␮g/ml) as loading controls. Colloidal gold
staining (Pierce) of the full membrane was also used to evaluate loading
control.
Coronin-1 detection by indirect immunofluorescence and
immunocytochemistry
PLB985 cells were cytocentrifuged and labeled as previously described
(30). Briefly, cells were fixed with 3.7% formaldehyde and permeabilized
with 0.25% Triton X-100. They were immunolabeled with the mouse
monoclonal anti-coronin-1 followed by an Alexa Fluor 594 anti-mouse IgG
(Molecular Probes). Lung explant specimens from four of the CF patients
were obtained at transplantation (Hôpital Européen Georges Pompidou).
Immunohistochemistry was performed on paraffin 5-␮m-thick tissue sections using the mouse polyclonal anti-coronin-1 Ab, diluted 1/50 or a the
mouse monoclonal anti-cytokeratin Ab, diluted 1/200 (Santa Cruz Biotechnology) and immunoperoxidase detection using EnVision Dual Link System-HRP (DakoCytomation), as recommended by the manufacturer. Examinations were performed under a photomicroscope (Leica DMRB).
Statistical analysis
Statistical analysis was performed with Statview software for Windows XP
Version 3.1 with a one-way ANOVA. Statistical significance was established at p ⬍ 0.05.
Results
Proteomic analysis of neutrophil cytosolic proteins under basal
and apoptotic conditions
Induction of physiological apoptosis was confirmed by the marked
increase of phosphatidylserine externalization measured by flow
cytometry after PE-annexin-V labeling. (Fig. 1A): 9.6 ⫾ 0.6% before induction vs 71.1 ⫾ 1.2% after (n ⫽ 29, p ⬍ 0.001). Note
almost total absence of 7-AAD labeling, which confirms the absence of cell necrosis. Apoptosis was also confirmed by Western
blot analysis of caspase-3, showing the cleavage of the procaspase-3 (at 37 kDa under basal conditions) into its 17-kDa activation fragment (Fig. 1B) and by measuring mitochondrial depolarization after DIOC6 labeling (data not shown). Next,
neutrophil cytosolic proteins under basal or apoptotic conditions
were analyzed. Technical reproducibility was deemed satisfactory
because the matching of spots between two separate 2D gels of the
same sample and were 78 and 76% under basal and apoptotic
conditions, respectively. Visual and computerized screening of the
maps obtained under basal or apoptotic conditions were analyzed
to identify proteins displaying the greatest variability between the
two states. Six spots could clearly be identified as sharply diminished under apoptotic compared with basal conditions, and five
spots were seen only under apoptotic conditions. These 11 spots
were excised from the gels, processed, and analyzed by MALDITOF mass spectrometry for protein identification (Table I). The
spots whose intensity was decreased during apoptosis were fulllength p38MAPK (Fig. 1C), gelsolin (Fig. 1E), moesin (Fig. 1G),
and coronin-1. The spots whose intensity was increased during
apoptosis were identified as fragments of coronin-1 and pyruvate
kinase. Both full-length and a cleavage fragment were identified
for annexin-1 as spots that were larger in the apoptotic state. Most
of the highly abundant cytosolic proteins that varied widely during
apoptosis were cytoskeleton-associated proteins including gelsolin, moesin and coronin-1, which are modified during apoptosis. A
strong p38MAPK decrease was also observed. These observations
were confirmed by immunoblot analysis of isolated neutrophils
from three different blood donors, using specific Abs against
p38MAPK (Fig. 1D), gelsolin (Fig. 1F), and moesin (Fig. 1H).
Western blot analysis of gelsolin showed several cleavage
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HEPES (50 mM (pH 7.4)), supplemented with 400 ␮M leupeptin (SigmaAldrich), 400 ␮M pepstatin, 4 mM PMSF, and 2 mM EDTA. After centrifugation for 15 min at 10,000 ⫻ g and 4°C, the protein concentration was
quantified using the BSA protein-assay kit (Pierce). Finally, proteins were
solubilized in 8 M urea, 2.5 M thiourea, 4% CHAPS w/v, 50 mM DTT
(Sigma-Aldrich), protease inhibitors (Bio-Rad), 1 mM orthovanadate, and
0.5% ampholytes (pH 4 –7 or 3–10). Linear (18 cm; pH 4 –7 or 3–1 0) IPG
strips (GE Healthcare) were loaded with 200 ␮g of proteins by passive
overnight rehydration. Isoelectrofocusing was run on IPGphor for 60,000
V-h. Focused strips were reduced and alkylated by incubating for 20 min
in 30% (v/v) glycerol, 2% SDS w/v, 6 M urea, and 50 mM Tris-HCl (pH
6.8) (equilibration buffer) containing 2% DTT, and then in equilibration
buffer containing 4.5% iodoacetamide. Strips were then placed on top of
10% polyacrylamide gels and run on the PROTEAN R II XL (Bio-Rad)
system. The gels were subsequently stained with standard ammoniacal
stain for differential gel analysis. The MS-compatible silver stain was used
for MS experiments (27). Technical reproducibility was evaluated using
Image Master Platinum software (version 5.00). Protein spots were excised
from the SDS-PAGE, processed, digested with trypsin (Promega) and
loaded on MALDI target, according to previously described procedures
(28). Mass spectrometry spectra, obtained with Bruker Autoflex mass spectrometer (Bruker-Daltonik) in reflector mode, were processed manually
using Bruker’s FlexAnalysis software, version 2.0. Peptide mass fingerprinting was performed using the MASCOT in-house server. Protein
searches were conducted in the SwissProt and NCBInr databases.
The Journal of Immunology
products ⬃60 kDa, which were not identified by mass spectrometry probably because they were not abundant enough (Fig.
1F). Same remark was also valid for moesin, which was cleaved
during apoptosis with the appearance of several cleavage products (Fig. 1H).
Evidence of coronin-1 cleavage during neutrophil apoptosis
Unlike the other cytoskeleton-associated proteins that we identified, coronin-1 has not yet been implicated in neutrophil apoptosis.
FIGURE 3. Characterization of PLB985 stably expressing coronin-1. A,
Western blot analysis of PLB985 cells using anti-HA Ab. The HA-tag was
inserted either at the carboxyl-terminal (PLBCoroCter) or amino-terminal
(PLBCoroNter) end of coronin-1. Recombinant HA-tagged-coronin-1 expression was detected in both PLBCoroCter and PLBCoroNter, whereas no
expression was detected in PLBpcDNA3 transfected with an empty plasmid or in nontransfected PLB985. Actin expression served as loading control. B, Western blot analysis of PLB985 using anti-coronin-1 Ab showing
the markedly enhanced expression of coronin-1 in both PLBCoroCter and
PLBCoroNter compared with that in PLBpcDNA3 or PLB985, containing
only endogenous coronin-1. Actin expression served as loading control. C,
Indirect immunofluorescence using anti-coronin-1 Ab showed more coronin-1 expression in PLBCoroCter and PLBCoroNter than PLBpCDNA3.
Nuclear chromatin was visualized by Hoechst staining. D, Flow cytometry
analysis of immunolabeled CD11b expression on PLB985 before and after
DMF-induced granulocyte differentiation. As expected, DMF induced a
dramatic increase in CD11b expression indicative of granulocytic differentiation (p ⬍ 0.001 using ANOVA). The data represent the means ⫾ SEM
of six different experiments. Coronin-1 Western blot analysis performed on
PLBpCDNA3, PLBCoroCter, and PLBCoroNter showed an increased coronin-1 expression in PLB985 cells after DMF-induced differentiation.
Colloidal gold staining of the membrane allows to show equal loading in
all lanes and was used as a loading control.
On 2D gel analysis, the coronin-1 fragment appeared at 30 kDa
and at an isoelectric point (pI) of 6 and the theoretical full-length
coronin-1 pI is 6.25 (Fig. 2A). Western blot analysis of coronin-1
in apoptotic neutrophils confirmed its cleavage into a 30-kDa fragment, which corresponds to the fragment size identified by mass
spectrometry (Fig. 2B). Using caspase-specific inhibitors, we observed that coronin-1 cleavage was inhibited by the pancaspase
inhibitor Z-VAD-FMK and that this inhibition was dose dependent. Accordingly, inhibition of coronin-1 cleavage by Z-VADFMK was associated with a decreased neutrophil apoptosis as
measured by annexin-V labeling with a minimum of 7-AAD-labeled neutrophils (⬍5%) (Fig. 2B). In contrast, apoptosis-induced
coronin-1 cleavage was not inhibited by Z-DEVDL-D-FMK, a
caspase-3/7 inhibitor, nor by Z-LEHD-FMK, a caspase-9 inhibitor,
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 2. Identification of a coronin-1 cleavage product during apoptosis. A, Fragment of coronin-1, identified at 30 kDa and pI of 6 on a 2D
gel, which appears under apoptotic conditions. The identification covers
26% with a score at 92 thus being fairly relevant because superior to 50. B,
Western blot analysis of coronin-1 in neutrophils during apoptosis and
effect of caspase inhibitors (10 ␮g/lane). Physiological apoptosis was induced in neutrophils by incubation at 37°C for 15 h in the absence (No) or
presence of either the pancaspase inhibitor Z-VAD-FMK (50 and 100 ␮M)
or the caspase-3 inhibitor Z-DEVDL-D-FMK (C3) (100 ␮M) or the
caspase-9 inhibitor, Z-LEHD-FMK (C9) (50 ␮M). Coronin-1 appears as a
doublet with the upper band at 57 kDa under basal conditions but is cleaved
into a 30-kDa fragment during apoptosis (Ap). Colloidal gold staining of
the membrane allows to show equal loading in all lanes and was used as a
loading control. Histograms show the percentages of annexin-V positive
and 7-AAD negative neutrophils to evaluate the effect of caspase inhibitors
on neutrophil apoptosis. Of note, Z-DEVDL-D-FMK and Z-LEHD-FMK
induced a high percentage of necrosis (⬎50% of 7-AAD-positive cells) at
200 and 100 ␮M respectively, data not shown). Data are means ⫾ SEM of
five independent experiments, ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 using ANOVA. C,
Western blot analysis of coronin-1 in neutrophils under basal (B) or apoptotic (Ap) conditions (5 ␮g/lane) to visualize the decrease in the fulllength coronin-1. Colloidal gold staining of the membrane allows to show
equal loading in all lanes and was used as a loading control. D, Western
blot analysis of coronin-1 in neutrophils treated with inflammatory cytokines (10 ␮g/lane). Neutrophils were treated for 4 h with either IL-8, GCSF, or TNF-␣ at the indicated concentrations expressed in nanograms per
milliliter, thus showing an increased coronin-1 expression after IL-8 and
G-CSF treatment. The arrow indicates the upper band of the doublet of
coronin-1, which appears at 57 kDa. Anti-actin Western blot analysis was
used as internal control loading. In each panel, A–C, the experiment shown
is representative of four different experiments in four different donors
which gave similar results.
7257
7258
thereby showing that coronin-1 was not a direct substrate of these
caspases. However, caspase-3 inhibitor treatment at 100 ␮M resulted in a modest but significant decrease in neutrophil apoptosis
(Fig. 2B). Of note, at 200 ␮M ⬎50% were necrotic and 7-AAD
positive, thus suggesting that the effect on neutrophil survival was
nonspecific. For the caspase-9 inhibitor, no decrease in neutrophil
apoptosis was observed between 1 and 50 ␮M, whereas at 100
␮M, ⬎50% of neutrophils were 7-AAD-positive neutrophils, thus
rendering the data difficult to interprete. The coronin-1 cleavage
was concomitant with the decrease in full-length coronin-1 as
demonstrated on the Western blot analysis (Fig. 2C). We concluded that apoptosis was associated with coronin-1 cleavage and
we next wanted to investigate if, conversely, neutrophil survival
induced by proinflammatory stimuli would up-regulate coronin-1
expression. Neutrophils were treated for 4 h with either IL-8 (50 –
100 ng/ml) or TNF-␣ (1–50 ng/ml) or G-CSF (1–50 ng/ml). We
have observed by Western blot analysis that coronin-1 expression
FIGURE 5. Coronin-1 expression decreased TRAIL-induced apoptosis. A, Analysis of TRAIL-induced apoptosis in PLBCoroCter and in
PLBCoroNter as compared with control PLBpcDNA3. TRAIL triggered a
significant increase in the percentages of cells showing mitochondrial depolarization in undifferentiated PLB985 (upper panel) and in DMF-differentiated cells (lower panel). B, Measurement of caspase-8 activity using
specific chromogenic substrate under basal conditions and after TRAILinduced apoptosis. C, Western blot analysis of Bid in control PLBpcDNA3
(lane 1) and in PLBCoroNter (lane 2) under basal conditions or after
TRAIL-induced apoptosis. Anti-actin was used as loading control. Measurement of caspase-9 (D) and caspase-3 (E) activities using specific chromogenic substrate under basal conditions and after TRAIL-induced apoptosis. Data presented in A, B, D, and E are means ⫾ SEM of six
independent experiments, ⴱp ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 using ANOVA. Significant difference between nontreated and gliotoxin-treated cells are indicated on the corresponding lines and comparison between control
PLBpcDNA with PLBCoroCter or PLBCoroNter are indicated by ⴱ§.
was increased by IL-8 and by G-CSF but not by TNF-␣ (Fig. 2D).
Interestingly, both IL-8 and G-CSF increased neutrophil survival
(as evidence by a significant decreased percentages of cells showing annexin-V-positive labeling as compared with controls, data
not shown) after an overnight incubation at 37°C. In contrast,
TNF-␣ (at the concentrations that we have tested) did not have any
effect on neutrophil survival. Taken together, our present data
strongly suggest that coronin-1 can be induced by inflammatory
stimuli and that this induction correlated with neutrophil survival,
thus confirming that coronin-1 might be considered as a prosurvival factor induced by inflammation.
Functional analysis of coronin-1 in PLB985 cells stably
transfected with coronin-1
To investigate whether coronin-1 has a direct role in the modulation of neutrophil activation and survival, we used a gain of function approach based on the overexpression of coronin-1 in PLB985
promyelocytic cells, which can differentiate into mature neutrophils and constitute a model of surrogate neutrophils. PLB985 stably transfected with the cDNA coding for HA-tagged coronin-1
either at the amino-terminal (CoroNter) or at the carboxyl-terminal
(CoroCter), we selected those expressing the transgene by their
neomycin resistance to obtain the PLBCoroNter and PLBCoroCter, respectively.
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FIGURE 4. Less apoptosis-induced mitochondrial depolarization and
chromatin condensation in gliotoxin-treated PLB985 overexpressing coronin-1 as compared with controls. A, Flow cytometry analysis of mitochondrial depolarization measured in control PLBpcDNA3, in PLBCoroCter
and in PLBCoroNter after DIOC6 labeling under basal conditions (thin
lines) and after gliotoxin-induced apoptosis (bold lines) in undifferentiated
PLB985 (left panels) and in DMF-differentiated cells (right panels). This
experiment is representative of six independent experiments yielding similar results. B, Comparisons of the percentages of cells showing mitochondrial depolarization after gliotoxin-induced apoptosis. Data are means ⫾
SEM of six independent experiments, ⴱ, p ⬍ 0.05; and ⴱⴱ, p ⬍ 0.01 using
ANOVA. Significant difference between nontreated and gliotoxin-treated
cells are indicated on the corresponding lines and comparison between
control PLBpcDNA with PLBCoroCter or PLBCoroNter are indicated by
ⴱ§. C, Microscope observation of chromatin condensation with or without
gliotoxin. Without gliotoxin, the nuclear appearance in the three cell types
were similar, whereas after gliotoxin addition, the number of cells showing
characteristic punctated nuclei decreased sharply and most radically in
PLB985pCDNA. D, Percentages of cells showing the punctated nuclear
morphology characteristic of apoptosis. Data are means ⫾ SEM of four
independent experiments (ⴱⴱ, p ⬍ 0.01 and ⴱ, p ⬍ 0.05 using ANOVA).
Significant difference between nontreated and gliotoxin-treated cells are
indicated on the corresponding lines and comparaison between control
PLBpcDNA with PLBCoroCter or PLBCoroNter are indicated by ⴱ§.
ANTIAPOPTOTIC EFFECT OF CORONIN-1 IN NEUTROPHILS
The Journal of Immunology
7259
Expression of recombinant coronin-1 in PLBCoroNter and
PLBCoroCter was verified by immunoblot analysis using an antiHA Ab to detect recombinant coronin-1 expression, whereas control PLB985 or PLB985pcDNA transfected with the empty plasmid were not reactive (Fig. 3A). The higher coronin-1 content
(including endogenous and recombinant coronin-1) in both PLBCoroNter and PLBCoroCter as compared with controls was confirmed by immunoblot analysis using a mouse monoclonal anticoronin-1 Ab (Fig. 3B). Indirect immunolabeling of coronin-1
confirmed more coronin-1 expression in PLBCoroNter and PLBCoroCter than PLBpcDNA (Fig. 3C).
To determine whether coronin-1 overexpression could modify
DMF-induced granulocyte differentiation, membrane expression
of CD11b, which is a marker of mature neutrophils was evaluated.
After 5 days of DMF exposure, PLB985 exhibited a significant
increase in the percentage of cells expressing CD11b as compared
with untreated cells ( p ⬍ 0.01) (Fig. 3D) and the characteristic
morphological features of mature neutrophils assessed by May-
Gruenwald staining (data not shown). Coronin-1 overexpression
did not affect the kinetics of granulocyte differentiation, because
the percentages of CD11b-positive cells were similar in coronin1-expressing cells and in control transfected cells. Interestingly,
Western blot analysis of coronin-1 in PLB985 before and after
DMF-induced differentiation showed that coronin-1 expression
was increased after DMF-induced differentiation in all three cell
lines either control, or coronin-1-transfected PLB985. Taking into
account the role of coronin-1 in phagosome fusion, coronin-1 is
part of the effector mechanisms that mature neutrophils will use
when challenged with microorganisms.
We next examined whether coronin-1 overexpression could
modulate PLB985 apoptosis. Apoptosis was induced by gliotoxin,
which triggers mitochondria depolarization, a typical feature of
apoptotic death (23, 24). Mitochondria depolarization was evaluated by flow cytometry as the percentage of cells showing a decreased DIOC6 labeling (Fig. 4A). Gliotoxin-induced apoptosis
was decreased in coronin-1 overexpressing PLB985 after
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FIGURE 6. Neutrophils from CF patients have a delayed apoptosis and express more coronin-1. A, Apoptosis-induced phosphatidylserine externalization
on neutrophils from controls (n ⫽ 29) (䡺) and from CF patients (n ⫽ 27) (f). Neutrophils were incubated at 37°C and tested at different times for
annexin-V labeling. Data are the means ⫾ SEM, ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 using ANOVA. B, Representative experiment showing DIOC6 labeling in
neutrophils from one CF patient (in gray) as compared with a control donor (D) (white) to evaluate mitochondria depolarization after a 6-h incubation at
37°C. In the control donor, a peak with lower fluorescence is indicative of mitochondria depolarization, whereas no such peak was detectable in neutrophils
from CF patients. C, Apoptosis-induced mitochondria depolarization was evaluated by the percentage of neutrophils within the peak of lower fluorescence
in control donors (n ⫽ 10) (䡺) and in CF patients (n ⫽ 12) (f). Data are the means ⫾ SEM, ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 using ANOVA. D, Representative
experiment showing a 2D electrophoresis proteomic map of neutrophil cytosolic proteins under basal conditions from a control donor and a CF patient.
The circled protein overexpressed in the CF patient has been identified as full-length coronin-1. E, Western blot analysis of coronin-1 in neutrophils from
the same control donor (D) and CF patient shown in D. The percentage of annexin-V-positive neutrophils after 4-h incubation at 37°C were 65.2 and 11.3%
for the control donor and the CF patient, respectively, thus confirming the increased survival capacity of neutrophils from CF patients. GAPDH served as
a loading control.
7260
Increased coronin-1 expression in neutrophils from CF patients
Then, the potential clinical relevance of the antiapoptotic activity
of coronin-1 was explored using neutrophil from CF patients. Kinetic analysis of phosphatidylserine externalization showed that
CF patients’ neutrophils underwent significantly less apoptosis and
occurred later and more slowly than controls at all time points
tested. For instance, after a 6-h incubation, the mean percentages
of annexin-V-positive CF and normal neutrophils were 51.55 ⫾
2.7% on 34.67 ⫾ 2.83%, respectively ( p ⬍ 0.01) (Fig. 6A). Likewise, a significant delay in mitochondria depolarization measured
after DIOC6 labeling was also observed (Fig. 6, B and C). No
relationship was found between the patient’s age and the apoptosis
rate, thereby excluding an age effect in CF neutrophils. Proteomic
analysis of cytosolic proteins from neutrophils freshly isolated
FIGURE 7. Coronin-1 expression in neutrophils within airways in lung
tissues from a CF patient. Immunoperoxidase labeling of coronin-1 in lung
explants with mouse monoclonal anti-coronin-1 Ab (A, C, and D) and with
anti-cytokeratin (B). A, Bronchial wall showing numerous coronin-1-labeled neutrophils in the lumen (arrow) in mucosa (circled) and in the lamina propria at low magnification (⫻40). B, Immunolabeling of bronchial
epithelial cells with a mouse monoclonal anti-cytokeratin (Œ). Note the
total absence of labeling in neutrophils (arrow), thus precluding any artifactual staining due to neutrophil endogenous peroxidase activity. C, High
magnification (⫻100) of the lumen content showing neutrophils expressing
coronin-1. D, High magnification of coronin-1-expressing neutrophils on
the lamina propria (circled), notably within a vessel (arrow). Similar observations were made in lung explants from four CF patients.
from CF patients compared with control neutrophils was conducted to identify proteins involved in CF neutrophil survival. As
shown on the 2D gels of neutrophil cytosolic proteins under the
basal conditions, the contents of several proteins were different.
Visual screening identified coronin-1 as a 57-kDa protein (at the
theoretical pI of 6.25) overexpressed in the CF patients’ cells, as
compared with control cells (Fig. 6D). Western blot analysis confirmed the significantly higher coronin-1 expression in CF neutrophils than control cells (Fig. 6E). This increased coronin-1 expression was confirmed by Western blot analysis in neutrophils from
seven CF patients showing a delayed apoptosis rate (data not
shown). Because coronin-1 could be considered a prosurvival factor in neutrophils and thus potentially inflammatory, we evaluated
whether coronin-1 was expressed in inflammatory neutrophils
from airways, thereby participating in prolonging CF neutrophil
survival at the site of pulmonary inflammation. To do so, coronin-1
expression was investigated by immunohistochemistry of pulmonary tissues obtained from five CF patients during transplantation
(Fig. 7). Neutrophils, which represent ⬎90% of the cells in the
bronchial lumen, strongly express coronin-1 in their cytoplasm
(Fig. 7A). At higher magnification, an heterogeneity of coronin-1
expression between neutrophils could be observed mostly likely
reflecting the survival status of the neutrophils (Fig. 7B). Likewise,
the cytoplasm of neutrophils present within the mucosa, lamina
propria and vessels (Fig. 7D) were also strongly labeled. This
strong coronin-1 expression in neutrophils contrasts with the lack
of labeling observed in cells present in the lamina propria, including fibroblasts and lymphocytes (Fig. 7D). Immunolabeling of cytokeratin restricted to epithelial cells, as expected, provided evidence that coronin-1 labeling was not due to neutrophil
endogenous peroxidase (Fig. 7B). These observations suggest that
coronin-1 might play a role in neutrophil survival at the site of
inflammation.
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DMF-induced differentiation ( p ⬍ 0.01), compared with controls
(Fig. 4B), whereas this decrease was not statistically significant in
undifferentiated cells. Apoptosis was also evaluated as the percentage of cells showing chromatin condensation after Hoechst staining (Fig. 4C). Gliotoxin triggered apoptosis of undifferentiated
PLB985 but no apoptosis was detected under basal conditions.
Interestingly, significantly lower percentages of coronin-1-expressing
PLB985cells contained condensed chromatin as compared with controls (Fig. 4D). Likewise, the percentages of DMF-differentiated coronin-1-expressing PLB985 undergoing apoptosis was lower than
controls. The HA-tag position in the recombinant coronin-1 had no
effect because coronin-1 induced antiapoptotic effect, as assessed by
chromatin condensation or by mitochondrial depolarization, was observed in both PLBCoroNter and PLBCoroCter.
We then wondered whether coronin-1 could also modulate the
extrinsic death receptor-dependent apoptotis pathway, particularly
the TRAIL-induced pathway. TRAIL-induced mitochondrial depolarization was significantly inhibited both in PLBCoroCter and
in PLBCoroNter after differentiation as compared with controls,
thus suggesting that coronin-1 can also modulate apoptosis via the
extrinsic pathway (Fig. 5A). Of note, we confirmed that the spontaneous apoptosis of DMF-differentiated PLB985 observed in the
absence of TRAIL was significantly decreased in coronin-1-expressing cells. We first assessed the activity of the initiator
caspase-8 that is activated after death receptor clustering by
TRAIL or during spontaneous neutrophil apoptosis. Caspase-8
triggers mitochondria depolarization via the cleavage of Bid, a
pro-apoptotic Bcl-2 homologue. As expected, caspase-8 activity
assessed by spectrophotometry using a chromogenic substrate was
significantly increased by TRAIL in PLBCoroCter, PLBCoroNter,
and in the control PLBpcDNA. However, no difference in TRAILinduced caspase-8 activity was observed between coronin-1-expressing PLB985 and controls (Fig. 5B). Accordingly, TRAIL decreased Bid expression because of its cleavage as shown by
Western blot analysis of Bid expression both in coronin-1-expressing PLB985 and in controls (Fig. 5C), thus strongly suggesting that
coronin-1 did not interfere upstream the mitochondria level in
TRAIL-induced apoptosis. We next investigated caspase-9 activation that occurs after the release of proapoptotic factors from the
mitochondria and caspase-3 activation downstream of both initiators caspases, caspase-8 and caspase-9. In cells subjected to
TRAIL-induced apoptosis, significantly less caspase-9 (Fig. 5D)
and caspase-3 (Fig. 5E) activities were observed in coronin-1overexpressing cells compared with controls. Taken together,
these data led us to conclude that coronin-1 inhibits apoptosis
in the surrogate neutrophil model of DMF-differentiated PLB985.
This inhibition occurred upstream from mitochondrial depolarization both in the receptor-dependent and in the mitochondria-dependent pathways.
ANTIAPOPTOTIC EFFECT OF CORONIN-1 IN NEUTROPHILS
The Journal of Immunology
Discussion
(38). Interestingly, coronin-1 copurified with a complex containing
the proteins p67phox, p47phox and p40phox, which are the cytosolic components of the membrane-bound NADPH oxidase system
(40). Binding studies provided evidence for a direct interaction
between coronin-1 and the carboxyl-terminal half of p40phox (18).
p40phox is constitutively associated with p67phox via a high affinity interaction. It has been recently identified a positive role for
p40phox in coupling NADPH oxidase activation to Fc␥IIA receptor-induced phagocytosis (41), but the exact role of coronin-1 in
regulating the NADPH oxidase remains to be elucidated.
The major finding of this study is the antiapoptotic role of coronin-1 in DMF-differentiated PLB985 cells. We observed that
coronin-1 expression was increased after DMF-induced differentiation, thus suggesting that it might be an important element in the
survival mechanisms of mature neutrophils. Indeed, coronin-1
overexpression significantly decreased apoptosis triggered either
by gliotoxin, a pharmacologic agent described as a bak agonist, or
by TRAIL acting via the death receptor pathway. Mitochondrial
dysfunction, an early event preceding nuclear and plasma membrane changes, is characterized by an enhanced mitochondrial
membrane permeability and loss of membrane potential, resulting
in the release of cytochrome c into the cytosol. Once released,
cytochrome c binds to apoptotic protease-activating factor-1 in the
presence of ATP and forms a complex that processes and activates
procaspase-9, which, in turn, cleaves and activates the executioner
caspases, e.g., caspase-3 and caspase-7. Coronin-1 inhibition of
both TRAIL- or gliotoxin-induced apoptosis suggests a common
point in the apoptotic pathway, which is consistent with a role at
the mitochondrial step. Moreover, in TRAIL-induced apoptosis,
we could demonstrate that coronin-1 overexpression does not affect Bid cleavage nor caspase-8 activity, whereas significant decrease in caspase-9 and caspase-3 activities were observed. These
data strongly corroborate that coronin-1 acts at the mitochondrial
level even when the agonist stimulates the death receptor pathway.
It could be hypothesized that coronin-1 interferes with proapoptotic molecules, like bax or bak. However, elucidation of the molecular mechanisms governing coronin-1 antiapoptotic activity
will require further investigation and might involve specific partners of coronin-1 in relationships with the cytoskeleton. Moreover,
1) because NADPH oxidase activation is closely linked with apoptosis induction (5) and 2) because coronin-1 is a partner of
p40phox (18), it would be important to investigate whether this
antiapoptotic effect of coronin-1 could be linked with the NADPH
oxidase system.
In addition, it would be crucial to understand how coronin-1 is
regulated in neutrophils. Interestingly, we provide evidence that
coronin-1 expression is increased by IL-8 and G-CSF which concomitantly induce neutrophil survival. In contrast, TNF-␣, which
did not induce a prosurvival effect in our experiments when tested,
did not increase coronin-1 expression. We should extend our data
with inflammatory cytokines and prosurvival factors both at the
protein and at the mRNA levels (42, 43). The overall picture would
thus be that, in neutrophils, apoptosis triggered coronin-1 cleavage,
whereas survival is associated with an increase in coronin-1 expression which, itself, potentiates survival.
Recent data have implicated a coronin-1 role in T lymphocyte
survival in coronin-1-deficient mice (44). Those authors reported
higher percentages of annexin-V-positive CD4⫹ and CD8⫹ thymocytes and peripheral naive T cells. Increased apoptosis of coronin-1-deficient T cells was associated with higher spontaneous
release of cytochrome c into the cytoplasm and cleavage of
caspase-3 and caspase-9, which was reversed by caspase-specific
inhibitors (44). In another study, coronin-1 was also implicated in
T cell survival via the generation of inositol-1,4,5-triphosphate and
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Proteomic approaches were previously used to characterize the
proteins differentially expressed in neutrophils under inflammatory
conditions (31), those implicated in myeloid differentiation (32) or
granular proteins (33). Using a subcellular proteomic approach,
Burlak et al. (34) provided evidence that neutrophil phagosomes
contain proteasome, endoplasmic reticulum, and mitochondrial
components, which had not been observed before, thereby suggesting potential new phagosome functions beyond microbial killing. Our findings presented herein, confirmed that the proteomic
approach is a useful tool to investigate neutrophil apoptosis, which
might involve complex protein modifications and cell-specific protein cleavage. Over the past few years, studies performed on apoptotic cells identified several cytoskeleton proteins, e.g., moesin
or gelsolin, as caspase substrates (26). To the best of our knowledge, the results of our study are the first to demonstrate that coronin-1 was cleaved during neutrophil apoptosis and that coronin-1
overexpression is associated with an increased neutrophil survival.
We provided evidence that apoptosis-induced coronin-1 cleavage
was dependent on caspase activity because the pancaspase inhibitor Z-VAD-FMK could reverse apoptosis-induced coronin-1
cleavage. However, the lack of effect of Z-DEVDL-D-FMK, a
specific caspase-3 inhibitor on coronin-1 cleavage strongly suggested that coronin-1 was not a direct substrate of caspase-3. Likewise, Z-LEDH-D-VMK, a specific caspase-9 inhibitor did not totally reverse apoptosis-induced coronin-1 cleavage. In addition,
using an in vitro assay of procaspase-9 activation composed of
neutrophil cytosol in the presence of ATP and cytochrome c, we
failed to induce coronin-1 cleavage (data not shown). Indeed, in
contrast with gelsolin, which is a direct substrate for caspase-3 (35)
and which has been described in different cellular models to be an
antiapoptotic protein, coronin-1 sequence does not contain a
caspase-specific consensus cleavage site. It is possible that proteinases other than caspases could be involved in apoptosis-induced coronin-1 cleavage. For instance, earlier studies showed that
apoptosis-induced p38MAPK in neutrophils was dependent on
serine-proteinase activities (36). Our data demonstrate that coronin-1 cleavage is dependent on apoptosis and strongly suggest that
caspase activity might be involved. Further studies will thus be
required to fully characterize the molecular mechanisms involved
in apoptosis-induced coronin-1 cleavage in neutrophils.
We next examined whether coronin-1, like gelsolin (37), could
be considered by itself, as a survival factor and prevent apoptosis.
So far, no data were reported on a role of coronin-1 in the regulation of neutrophil apoptosis. Neutrophils express at the mRNA
level five of the seven human coronin genes namely coronins 1, 2,
3, 4, and 7 but not coronins 5 and 6 (38). However, most of the
studies on neutrophils focused on coronin-1. Dictyostelium coronin
(which is similar to coronin-1) appears to be associated to some
extent with all structures containing F-actin and is involved in
phagocytosis, macropinocytosis, cell locomotion, and cytokinesis.
Using coronin-GFP to investigate coronin localization and dynamics during actin-mediated endocytosis, it was shown that the characteristic “crowns” are macropinocytosis sites and that similar
structures are involved in phagocytosis (39). Results of studies on
human neutrophils suggested that the coronin-1 is associated with
endocytosis and has been conserved throughout evolution. Indeed,
coronin-1 was shown to accumulate with F-actin around endocytic
vesicles. In neutrophils, using a dominant-negative TAT-WD coronin-1, generated by the transduction of a coronin-1 domain containing the WD-repeat sequences and fused with a membrane permeable TAT transduction domain, it has been shown that
coronin-1 was involved in adhesion, spreading, and phagocytosis
7261
7262
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7.
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10.
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14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
Acknowledgments
We thank Dr. Philippe de Villartay for providing clinical data on CF patients as well as Valérie Gausson and Joyce Benchitrit for excellent technical assistance in neutrophil experiments. The expertise of Sophie Berissi
in immunohistochemistry (Histochemistry Facilities, Institut National de la
Santé et de la Recherche Médicale Unité 845) and the technical assistance
of Sébastien Roux for immunolabeling were greatly appreciated.
Disclosures
24.
25.
26.
The authors have no financial conflict of interest.
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calcium mobilization after TCR triggering (45). The role of coronin-1 in leukocytes, especially neutrophils, is still unclear, although it seems to be involved in multiple vital mechanisms, including modulation to cell survival.
Proteomic analysis of neutrophil cytosolic proteins has proven
to be highly informative when investigating CF patients’ neutrophils showing prolonged survival, attributed to delayed physiological apoptosis. It was previously demonstrated that CF neutrophils
from patients have lower Bax levels, thus potentiating their survival (11, 12). Indeed, several proteins were differently expressed
between freshly isolated neutrophils from a control and a CF patient. It is important to emphasize that coronin-1 was identified on
2D gels by MALDI-TOF mass spectra as a protein that was obviously overexpressed in cytosol of CF neutrophils under basal
conditions as compared with controls, thus validating the proteomic approach. Western blotting also confirmed that coronin-1
was overexpressed in circulating neutrophils from CF patients as
compared with controls, which is compatible with the delayed mitochondria depolarization that we have observed in neutrophils
from CF. In addition, coronin-1 was strongly expressed in neutrophils from CF patients’ bronchial airways, thus suggesting that
coronin-1 might be involved in the mechanisms leading to neutrophil persistence within CF airways. As a consequence of these
findings, it will now be relevant to study the regulation of coronin-1 expression in CF neutrophils to determine the molecular
mechanisms involved in this overexpression. However, most
likely, in CF neutrophils there might be more disturbances than a
single increase in coronin-1 levels and there should be several
“survival” pathways that are activated. Despite some controversies
concerning the functional expression of CFTR gene in neutrophils,
its encoded protein has been detected by Western blotting raising
the possibility that some inflammatory features of neutrophils
could result from the genetic defect (46). Using a gene microarray
approach, it has been recently shown that neutrophils from clinically stable and noninfected CF patients exhibited a different pattern of gene expression than those from controls (47). That difference was greater when comparing controls and CF patients’
“resting” neutrophils, than when comparing the latter and “inflammatory” neutrophils, isolated from bronchoalveolar lavages fluids.
These findings are in agreement with a perturbed “inflammatory
program” in CF neutrophils which remains to be investigated further to explore its possible modulation. Indeed, it becomes clear
that neutrophils could constitute a relevant cellular target in CF.
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