Cross-linking of human FcgammaRIIIb induces the production of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor by polymorphonuclear neutrophils.
ABSTRACT We have reported that human autoantibodies reacting with the polymorphonuclear neutrophil (PMN)-anchored FcgammaRIIIb (CD16) protect these cells from spontaneous apoptosis. In this study, we used anti-CD16 F(ab')(2) to delineate the mechanism(s) whereby the PMN life span is extended. As documented using four methods, CD16 cross-linking impeded spontaneous apoptosis, whereas anti-CD18 F(ab')(2) exerted no effect. Incubation of PMNs with anti-CD16 prevented the up-regulation of beta(2) integrins, particularly CD11b, which is the alpha-chain of complement receptor type 3, but also CD18, which is its beta-chain, as well as CD11a and CD11c. Anti-CD16-conditioned supernatant of PMNs diminished the percentage of annexin V-binding fresh PMNs after another 18 h in culture, whereas the negative control anti-CD18 had no effect. The expression of mRNA for G-CSF and GM-CSF was induced by anti-CD16, followed by the release of G-CSF and GM-CSF in a dose-dependent manner. Anti-G-CSF and anti-GM-CSF mAbs abrogated the antiapoptotic effect of the related growth factors. The delay in apoptosis was accompanied by a down-regulated expression of Bax, and a partial reduction of caspase-3 activity. These data suggest an autocrine involvement of anti-CD16-induced survival factors in the rescue of PMNs from spontaneous apoptosis. Thus, apoptosis of aged PMNs can be modulated by signaling through FcgammaRIIIb, which may occur in patients with PMN-binding anti-FcgammaRIIIb autoantibodies.
- SourceAvailable from: Christopher Haslett[show abstract] [hide abstract]
ABSTRACT: Mechanisms governing the normal resolution processes of inflammation are poorly understood, yet their elucidation may lead to a greater understanding of the pathogenesis of chronic inflammation. The removal of neutrophils and their potentially histotoxic contents is one prerequisite of resolution. Engulfment by macrophages is an important disposal route, and changes in the senescent neutrophil that are associated with their recognition by macrophages are the subject of this investigation. Over 24 h in culture an increasing proportion of human neutrophils from peripheral blood or acutely inflamed joints underwent morphological changes characteristic of programmed cell death or apoptosis. Time-related chromatin cleavage in an internucleosomal pattern indicative of the endogenous endonuclease activation associated with programmed cell death was also demonstrated. A close correlation was observed between the increasing properties of apoptosis in neutrophils and the degree of macrophage recognition of the aging neutrophil population, and a direct relationship between these parameters was confirmed within aged neutrophil populations separated by counterflow centrifugation into fractions with varying proportions of apoptosis. Macrophages from acutely inflamed joints preferentially ingested apoptotic neutrophils and histological evidence was presented for occurrence of the process in situ. Programmed cell death is a phenomenon of widespread biological importance and has not previously been described in a cell of the myeloid line. Because it leads to recognition of intact senescent neutrophils that have not necessarily disgorged their granule contents, these processes may represent a mechanism for the removal of neutrophils during inflammation that also serves to limit the degree of tissue injury.Journal of Clinical Investigation 04/1989; 83(3):865-75. · 12.81 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The last few years have seen the accumu- lation of compelling evidence that apoptosis (pro- grammed cell death) plays a major role in promoting resolution of the acute inflammatory response. Neutro- pulls are constitutively programmed to undergo apop- tosis, which limits their pro-inflammatory potential and leads to rapid, specific, and non-phlogistic recognitionKidney and Blood Pressure Research 02/2000; 23(3-5):173-4. · 1.60 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Human neutrophils constitutively undergo apoptosis and this process is critical for the resolution of inflammation. Whilst neutrophil apoptosis can be modulated by a wide variety of agents including GM-CSF, LPS and TNF-alpha, the molecular mechanisms underlying neutrophil death and survival remain largely undefined. Recent studies have shown the involvement of members of the Bcl-2 protein family (especially Mcl-1 and A1) and caspases in the regulation and execution of neutrophil apoptosis. Cell surface receptors and protein kinases, particularly mitogen-activated protein kinases, also play critical roles in transducing the signals that result in neutrophil apoptosis or extended survival. This review summarises current knowledge on the molecular mechanisms and components of neutrophil apoptosis.FEBS Letters 02/2001; 487(3):318-22. · 3.58 Impact Factor
of April 17, 2013.
This information is current as
Factor by Polymorphonuclear Neutrophils
Colony-Stimulating Factor and
the Production of Granulocyte
Cross-Linking of Human Fc
Pierre Youinou and Christophe Jamin
Véronique Durand, Yves Renaudineau, Jacques-Olivier Pers,
2001; 167:3996-4007; ;
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2001 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month by
The Journal of Immunology
by guest on April 17, 2013
Cross-Linking of Human Fc?RIIIb Induces the Production of
Granulocyte Colony-Stimulating Factor and
Granulocyte-Macrophage Colony-Stimulating Factor by
Ve ´ronique Durand,1Yves Renaudineau,1Jacques-Olivier Pers, Pierre Youinou,2and
We have reported that human autoantibodies reacting with the polymorphonuclear neutrophil (PMN)-anchored Fc?RIIIb (CD16)
protect these cells from spontaneous apoptosis. In this study, we used anti-CD16 F(ab?)2to delineate the mechanism(s) whereby
the PMN life span is extended. As documented using four methods, CD16 cross-linking impeded spontaneous apoptosis, whereas
anti-CD18 F(ab?)2exerted no effect. Incubation of PMNs with anti-CD16 prevented the up-regulation of ?2integrins, particularly
CD11b, which is the ?-chain of complement receptor type 3, but also CD18, which is its ?-chain, as well as CD11a and CD11c.
Anti-CD16-conditioned supernatant of PMNs diminished the percentage of annexin V-binding fresh PMNs after another 18 h in
culture, whereas the negative control anti-CD18 had no effect. The expression of mRNA for G-CSF and GM-CSF was induced by
anti-CD16, followed by the release of G-CSF and GM-CSF in a dose-dependent manner. Anti-G-CSF and anti-GM-CSF mAbs
abrogated the antiapoptotic effect of the related growth factors. The delay in apoptosis was accompanied by a down-regulated
expression of Bax, and a partial reduction of caspase-3 activity. These data suggest an autocrine involvement of anti-CD16-induced
survival factors in the rescue of PMNs from spontaneous apoptosis. Thus, apoptosis of aged PMNs can be modulated by signaling
through Fc?RIIIb, which may occur in patients with PMN-binding anti-Fc?RIIIb autoantibodies. The Journal of Immunology,
2001, 167: 3996–4007.
event sets off a swift uptake of aged PMNs by scavenger cells to
prevent the release of histotoxic products into the extracellular
milieu (1). In diseased sites, removal of apoptotic PMNs helps
contain the inflammatory process, and thereby limit tissue damage
(2). However, PMN longevity can be modulated by a variety of
agents, most notably cytokines (reviewed in Ref. 3). For example,
PCD is promoted by TNF-? (4) and IL-6 (5), but delayed by IL-
1?, G-CSF, GM-CSF, IFN-? (6), and IL-8 (7). Regarding the in-
tracellular pathways involved in spontaneous apoptotic mecha-
nism, the inhibitory effect of Bcl-2 remains uncertain in PMNs (8),
although the balance between Bax (9) and Bcl-xLhas been shown
to regulate the machinery governing the activity of caspase-3 in
these cells (10).
The low affinity receptors for the Fc region of IgG, Fc?RIIIb
(CD16), and Fc?RIIa (CD32) are naturally expressed in PMNs,
he short lifetime of circulating polymorphonuclear neu-
trophils (PMN)3is determined by programmed cell death
(PCD), also referred to as apoptosis. This inescapable
unlike the high affinity receptor Fc?RI (CD64), in which the syn-
thesis may be induced by IFN-? (reviewed in Ref. 11). Autoanti-
bodies directed against CD16 have been described in autoimmune
mice (12) and patients (13). In addition, on the basis of subsequent
results obtained by indirect immunofluorescence and ELISA tests,
we have identified three populations of anti-CD16 autoantibodies,
recognizing either PMN-bound CD16, soluble CD16, or both (14).
PMN-binding Fc?RIIIb autoantibodies might possibly be impli-
cated in the fate of PMNs. This concept is supported by our pre-
liminary results that show that, among anti-CD16 autoantibodies,
some with specificity for PMN-bound Fc?RIIIb have the capacity
to rescue senescent PMNs from spontaneous apoptosis (15). In-
triguingly, soluble Fc?RIIIb produces a similar effect (16). These
observations imply that CD16 may perpetuate inflammation, fol-
lowing autoantibody cross-linking, through the production of
PMN-derived cytokines (reviewed in Ref. 17). However, relatively
little is known about the induction and the mechanism(s) of such
processes. Although Fc?RIIIb is GPI anchored to the cell mem-
brane (18), several groups have established surprisingly that it can
transduce signals by itself (19–21). In contrast, others have stated
that Fc?RIIIb must cooperate with one or several neighboring
transmembrane partners. This latter view is strengthened by the
demonstration that Fc?RIIIb works in concert with Fc?RIIa to
activate PMNs (22–24). Alternatively, Fc?RIIIb can interact with
?2integrins, most notably complement receptor (CR) type 3, as
indicated by cocapping experiments (25). This major adhesion
molecule of PMNs (26) is formed by an ?-chain (CD11b) nonco-
valently linked to a ?-chain (Mac-1, CD18), which is shared by
other integrins, CD11a/CD18 (LFA-1), CD11c/CD18 (CR4), and
CD11d/CD18. One practical problem is that a proportion of anti-
Fc?RIIIb autoantibody-containing sera is not uniquely specific for
Laboratory of Immunology, Institut de Synergie des Sciences et de la Sante ´, Brest
University Medical School, Brest, France
Received for publication December 27, 2000. Accepted for publication July 19, 2001.
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.
1V.D. and Y.R. contributed equally to this work.
2Address correspondence and reprint requests to Dr. Pierre Youinou, Laboratory of
Immunology, Brest University, Medical School, 5 av. Foch, F 29 609 Brest Cedex,
France. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; ABC, Ab-
binding capacity; CR, complement receptor; M?, macrophage; MFI, mean fluores-
cence intensity; PCD, programmed cell death; PI, propidium iodide; SF, survival
factor; CD62L, CD62 ligand.
Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00
by guest on April 17, 2013
Fc?RIIIb, inasmuch as autoantibodies from some of them react
with Fc?RIIIb, but also Fc?RII and/or Fc?RI (27). Therefore, it
remains unclear whether Fc?RIIIb autoantibody-containing sera
inhibit PMN apoptosis either through Fc?RIIIb cross-linking
alone, through Fc?RII ligation alone, or through collaboration be-
tween these two Fc?R.
In the present study, we have used anti-CD16 mAb F(ab?)2to
eliminate several artifacts, e.g., one population of autoantibodies
cross-reacting with CD16 and CD32, or the coexistence in a given
serum of one group of anti-CD16 autoantibodies and a second of
anti-CD32 autoantibodies. We confirm that cross-linking of CD16
retards apoptosis of PMNs, and show that the proportion of
CD11bdimPMNs is increased, relative to aged PMNs. The key
mechanism of the sustained longevity of PMNs seems to be the
CD16-induced production of cytokines. For the first time, we pro-
vide evidence for the anti-CD16 Ab-triggered transcription of
mRNA for G-CSF and GM-CSF, followed by the synthesis and
resulting release of these cytokines. Such a contention was con-
firmed by the blocking effect of anti-G-CSF and anti-GM-CSF
mAbs. Another consequence of the CD16 stimulation was the low-
ered level of Bax (a proapoptotic member of the Bcl-eg2 family),
and the subsequent reduction in the caspase-3 activity. Thus, a
resolution of the inflammation might, at least in part, be modulated
Materials and Methods
FITC mAbs to CD16 (clone 3G8), CD11b (clone Bear 1), CD4 (clone
13B8.2), CD8 (clone B9.11), CD15 (clone 80H5), CD18 (clone 7E4),
CD19 (clone J4.119), CD23 (clone 9P25), CD32 (clone 2E1), CD35 (clone
J3.D3), CD45 (clone J33), CD48 (clone J4-57), CD56 (clone N901), CD64
(clone 22), CD66b (clone 80H3), and to monomorphic determinants in
HLA class I molecules (clone B9.12.1); PE mAbs to CD11a (clone 25.3.1),
CD11b (clone Bear 1), CD11c (clone BU-15), and CD18, as well as un-
conjugated mAbs to CD16, CD11b (clone Bear 1), HLA-I, CD3 (clone
UCHT1), CD4, CD8, CD18, CD19, CD23, CD56, and CD64 were all
obtained from Beckman Coulter (Villepinte, France). FITC mAb to CD53
(clone MEM 53) was purchased from Interchim (Montluc ¸on, France), and
FITC mAbs to CD55 (clone BRIC 110) and to CD59 (clone MEM-43)
from Serotec (Oxford, U.K.). Additional PE anti-CD11b mAbs, clones 44
and 2LPM19c, were obtained from BD PharMingen (San Diego, CA) and
Dakopatts (Glostrup, Denmark), respectively. FITC anti-CD62 ligand
(CD62L) (clone TQ1) mAb, FITC and PE IgG1, IgG2a, and IgM isotype
controls were purchased from Beckman Coulter. FITC-labeled and uncon-
jugated sheep F(ab?)2anti-mouse IgG F(ab?)2were purchased from Jack-
son ImmunoResearch Laboratories (West Grove, PA). Rabbit polyclonal
anti-Bax F(ab?)2was obtained from Santa Cruz Biotechnology (Santa Cruz,
CA), and revealed by FITC goat F(ab?)2anti-rabbit F(ab?)2(Sigma, St.
Louis, MO). Unconjugated anti-TNF-?, anti-G-CSF, and anti-GM-CSF
goat polyclonal Abs were obtained from R&D Systems (Minneapolis,
MN). Anti-CD16, anti-CD18, anti-HLA-I, and anti-CD11b F(ab?)2were
prepared by pepsin digestion.
Blood samples were drawn from healthy nonsmoking volunteers into hep-
arinized Vacutainer tubes (BD Biosciences, Franklin Lakes, NJ). Five-
milliliter aliquots were collected to study unmanipulated cells. These were
immediately cooled to 4°C, washed three times in 125 mM NaCl, 10 mM
phosphate, 5 mM KCl, 5 mM glucose, 1.09 mM CaCl2and 1.62 mM
MgCl2, pH 7.35, and resuspended to the original volume. PMNs were
isolated from the rest of the blood samples by Dextran T500 (Pharmacia,
Uppsala, Sweden) sedimentation, followed by Ficoll-Hypaque density gra-
dient centrifugation (Eurobio, Les Ulis, France). Residual erythrocytes
were lysed with hypotonic buffer. To reduce macrophage (M?), eosinophil,
T, B, and NK cell contamination below 1% (28), the cell suspension was
incubated for 20 min in the presence of a mixture of anti-CD3, anti-CD4,
anti-CD8, anti-CD19, anti-CD23, anti-CD56, and anti-CD64 mAbs. After
three washes in PBS supplemented with 5% BSA, a negative selection was
performed using goat anti-mouse Ig Ab-coated magnetic microbeads (Bio-
Advance, Emerainville, France).
M? were prepared from 100 ml of blood obtained from the same donors
as the PMNs. PBMCs were allowed to adhere to polystyrene for 2 h at
37°C. Nonadherent cells were washed away, whereas the M? were har-
vested with 0.05% trypsin/0.02% EDTA, washed three times, and cultured
for another 4 h in the absence or the presence of 500 pg/ml IL-1? (Gen-
zyme, Cambridge, MA). B cells from a patient with chronic lymphocytic
leukemia were also purified by negative selection with magnetic beads,
following treatment of the PBL with a mixture of mAbs, as described
To cross-link mAb binding to PMNs, 96-well microtiter plates were coated
with 30 ?g/ml sheep F(ab?)2anti-mouse F(ab?)2by an initial 30-min in-
cubation at 37°C, and a second at 4°C overnight, followed by three washes
with RPMI 1640 medium. PMNs were then dispensed at 5 ? 105cells/
well, and cultured in 200 ?l RPMI 1640 medium (Life Technologies, Pais-
ley, Scotland) supplemented with 2.5% FCS, 2 mM L-glutamine (bio-
Me ´rieux, Lyon, France), 200 U/ml penicillin, and 500 ?g/ml streptomycin.
CD16 mAb F(ab?)2was added at a final concentration of 5 ?g/ml, unless
otherwise indicated. In selected experiments, the effect of anti-CD16
F(ab?)2was evaluated in the absence of the second-layer Ab. It was es-
sential (6, 28) to ensure that all the reagents used for PMN isolation and
culture were LPS free, as judged by a quantitative chromogenic Limulus
amebocyte lysate assay (BioWhittaker, Walkersville, MD). As an addi-
tional precaution, polymyxin B (Sigma) was added at 100 IU/ml to neu-
tralize undetectable endotoxin trace amounts.
Staining consisted of incubating 5 ? 105PMNs for 30 min on ice with 10
?l appropriate mAbs at previously determined concentrations. After three
washes in PBS supplemented with 2% BSA and 0.1% sodium azide, PMNs
were analyzed on an Epics Elite flow cytometer (Coulter, Hialeah, FL),
along with isotype controls. The PMNs in whole blood were identified by
forward and side scatters. Purified PMNs were also gated to avoid un-
wanted debris. As for the anti-CD11b mAb, the binding of Bear 1 was not
blocked by either of the two I domain-specific CD11b mAbs, 44 and
2LPM19c (data not shown). The percentages of positive cells and mean
fluorescence intensities (MFI) were compared with isotype controls in all
experiments. In a series of experiments, saturation by Abs was investigated
by incubating 105freshly isolated PMNs with increasing amounts of un-
conjugated anti-CD16, anti-CD18, or anti-HLA-I F(ab?)2, and developing
with FITC-labeled sheep F(ab?)2anti-mouse IgG F(ab?)2. The MFI of the
second-layer reagent alone was systematically subtracted from that ob-
tained with previous incubation of anti-CD16, anti-CD18, or anti-HLA-I
F(ab?)2. In pilot experiments, the number of molecules per cell was quan-
tified by determining the amount of Ab binding to the cells at saturating
concentrations, using the Quantum Simply Cellular kit (Flow Cytometry
Standards, San Juan, PR). According to the manufacturer’s instructions, 50
?l of microbeads was added to 20 ?l of Ab. Ab-binding capacities (ABC),
derived from the MFI and accounting for the numbers of molecules rec-
ognized by these Abs, were expressed as arbitrary units.
Assessment of apoptosis
To determine apoptosis, 5 ? 105PMNs were stained with FITC-annexin
(29) and propidium iodide (PI), according to the manufacturer’s instruc-
tions (Beckman Coulter). After a 10-min incubation in the dark, cells were
analyzed by FACS. Early apoptotic PMNs were defined as those annexin
V?/PI?, and necrotic PMNs as PI?nonpermeabilized cells.
Independent assessment of apoptosis was evaluated using the method
described by Nicoletti et al. (30). Briefly, after 18 h in culture, PMNs were
washed in citrate buffer (0.1 M sodium citrate, 0.1% Triton X-100) and
incubated in 250 ?l of citrate buffer overnight in the dark at 4°C. Apoptosis
was expressed as the percentage of hypoploid PMNs in each flow cyto-
To confirm DNA fragmentation, PMNs were washed three times in
HBSS (Eurobio) and once in lysis buffer (250 mM sucrose, 50 mM Tris,
pH 7.5, 25 mM KCl, and 5 mM MgCl2). Cell pellets were resuspended and
incubated for 8 min on ice in a solution of 0.25% Triton X-100 (Sigma)
added to 500 ?l of lysis buffer. After centrifugation for 5 min at 500 ? g
and 4°C, nuclei were resuspended in 500 ml of lysis buffer. This was
supplemented with 25 ?l of 0.5 M EDTA, 70 ?l of 10% SDS, and 0.2 mg
of proteinase K (all obtained from Sigma), then incubated for 3 h at 37°C.
The DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:
1), followed by two extractions with chloroform (v/v). After 5 min of
washing at 400 ? g and 4°C, a 1:10 volume of sodium acetate, followed
by 2 vol of absolute ethanol was added. DNA was kept for 12 h at 4°C and
centrifuged for 10 min at 450 ? g. Two volumes of absolute ethanol were
3997The Journal of Immunology
by guest on April 17, 2013
then added before addition of 70% ethanol. DNA was dissolved in 10 mM
Tris, pH 7.5, and 1 mM EDTA, pH 8, for 12 h after evaporation of the
ethanol. The DNA was loaded into wells of a 1% agarose gel and electro-
phoresed at 75 mV using 100 mM Tris, 100 mM boric acid, and 0.2 mM
EDTA as running buffer. DNA was visualized by ethidium bromide
Cell morphology of 105fresh and apoptotic PMNs was studied. These
were centrifuged for 5 min at 200 ? g on microscope slides. The slides
were air dried for 10 min, and the cells were stained in a May-Gru ¨nwald
solution (Merck, Darmstadt, Germany) for 3 min, washed in water for 1
min, stained in a Giemsa solution (Merck) 7% in distilled water, and finally
rinsed in water. Morphologic changes characteristic of apoptosis, such as
nuclear condensation and vacuolation, were subsequently analyzed.
Identification of survival factors (SFs)
To address the question as to whether SFs are released by PMNs in re-
sponse to CD16 cross-linking, 5 ? 105PMNs were treated with anti-CD16,
anti-HLA-I, or anti-CD18 F(ab?)2at final concentrations of 1–20 ?g/ml.
After an 18-h incubation, supernatants were collected. These were incu-
bated with magnetic beads coated with anti-mouse F(ab?)2Ab for 30 min
to remove any residual F(ab?)2. Aliquots were then harvested to identify
SF(s), and the remaining 200 ?l, diluted 1/2 in fresh medium, was added
to triplicate wells of 5 ? 105freshly isolated PMNs. Incubation lasted a
further 18 h, until the time when annexin V?/PI?cells were enumerated in
these secondary cultures. Supernatant-induced variation of PCD was cal-
culated according to the formula: (control apoptotic PMNs ? supernatant
apoptotic PMNs/control apoptotic PMNs) ? 100.
RT-PCR of mRNA for SFs
Concomitantly, five different suspensions of 107fresh PMNs each were
cultured in the presence of 10 ?g/ml anti-CD16 F(ab?)2, and cells were
collected at 0, 3, 6, 9, and 12 h for RNA isolation. B cells, and resting or
IL-1?-activated M? served as control cells in the RT-PCR experiments.
For the preparation of RNA, PMNs, M?, or B cells were first washed twice
with HBSS. Total RNA was isolated by the guanidine isothiocyanate
method (31) using RNABle (Eurobio).
In the reverse-transcriptase step, 1 ?g of total RNA was used in a total
incubation volume of 20 ?l. Oligo(dT) primers, deoxynucleotides mix, and
200 U of reverse transcriptase from Moloney murine leukemia virus were
supplied (Life Technologies). After incubation at 42°C for 50 min, 2 U of
RNase H was added and incubated for 20 min at 37°C. Two microliters
from this incubation were used in each of the subsequent PCR amplifica-
Five pairs of primers were selected (32): 5?-CTCTGGACAGTGCAG
GAAGCCACC-3? plus 5?-GCTGGGCAAGGTGGCGTAGAACGC-3? for
G-CSF; 5?-GCAGCCCTGGGAGCATGTGAATGC-3? plus 5?-ATGCCT
GTATCAGGGTCAGTGTGC-3? for GM-CSF; 5?-AACTCCTTCTCCA
CAAGCGCCTTC-3? plus 5?-TGGACTGCAGGAACTCCTTAAAGC-3?
for IL-6; 5?-ACCGCCATGGAGGAAGGTCAATATTCAG-3? plus 5?-TA
TCCATGCTCAAGAGTGGAGAGGGGAG-3? for CD23; and 5?-GAAG
ATCAAGATCATTGCTCCTCC-3? plus 5?-CTGGTCTCAAGTCAGTGT
ACAGG-3? for ?-actin. DNA fragments of 533 bp (G-CSF), 497 bp (GM-
CSF), 593 bp (IL-6), 981 bp (CD23), and 707 bp (?-actin) were obtained.
The following program was used. After the initial template denaturation
for 5 min at 94°C, 2.5 U Taq polymerase (Genaxis Biotechnology, Saint-
Cloud, France) was added. Standard cycle conditions were: 30 s at 94°C,
60 s at 55°C, and 60 s at 72°C. Thirty-five cycles had to be conducted,
except for ?-actin, in which 30 cycles were sufficient. The resulting prod-
ucts were run on a 3% agarose gel (Nusieve 3.1; FMC, Rockland, ME) and
stained with 0.5 ?g/ml ethidium bromide. In addition, PCR products were
checked by digestion with various restriction enzymes. Expected digestion
patterns were obtained in each case. ?-actin mRNA was measured in RNA
samples at each point with the same cDNA as that analyzed for cytokine
transcripts. PCR band densities were determined using Molecular Analyst
software (Bio-Rad, Hercules, CA), and the mean density for each point was
normalized with ?-actin.
Synthesis and release of G-CSF and GM-CSF
G-CSF (sensitivity: 12 pg/ml) and GM-CSF (sensitivity: 5 pg/ml) were
assayed in the aliquots of CD16-conditioned medium, using commercial
ELISA kits (R&D Systems and Beckman Coulter, respectively). In inhi-
bition experiments designed to confirm the nature of SFs, human rG-CSF
and rGM-CSF (R&D Systems) were used to establish the specificity of the
related Abs by Western blotting (data not shown). Then, 200 ?l of the
primary culture supernatants was preincubated with 2 ?g of anti-TNF-?, 2
?g of anti-G-CSF, 2 ?g of anti-GM-CSF, or 1 ?g of anti-G-CSF plus 1 ?g
of anti-GM-CSF Abs for 90 min at 37°C, before being added to fresh
PMNs. Variation of spontaneous apoptosis was calculated as above.
Bcl-2 family member expression and caspase-3 activity
To identify intracellular Bax, PMNs were first labeled with PE anti-CD11b
mAb, and washed three times. They were then permeabilized in 1 ml of
0.1% saponin for 12 min in the dark. After two washes, the cells were
incubated with rabbit anti-Bax F(ab?)2for 30 min at 4°C, followed by
another three washes. This unconjugated Ab was revealed by a 30-min
incubation with FITC goat F(ab?)2anti-rabbit F(ab?)2at 4°C and washed
again. PMNs incubated with the second-layer Ab alone served as a nega-
tive control. The preparations were fixed in 5% paraformaldehyde buffer
before FACS analysis. A colorimetric assay was then used to measure
caspase-3 activity. As recommended by the manufacturer (Tebu, Le Per-
ray-en-Yvelines, France), 2 ? 107PMNs were lysed in 1 ml of lysis buffer
(50 mM HEPES, pH 7.4, 0.1% CHAPS, 1 mM DTT, 0.1 mM EDTA) for
5 min on ice. Cell extracts were mixed in assay buffer with or without
Ac-DEVD-CHO, a specific inhibitor for caspase-3 activity, in the presence
of Ac-DEVD-pNA substrate. Caspase-3 activity was measured at 405 nm
in an automatic plate reader (Multiskan Labsystems, Helsinki, Finland).
Means and SEM were calculated from a minimum of three independent
experiments, each run in triplicate (the results were averaged). Compari-
sons were made using the unpaired Mann-Whitney U test and the Wil-
coxon’s test for paired data.
Delayed spontaneous apoptosis of PMNs
The final cell suspension consisted of ?99% PMNs, as determined
by CD4, CD8, CD19, CD23, CD56, and CD64 staining (Fig. 1,
sedimentation, followed by Ficoll-Hypaque density gradient centrifuga-
tion. Upper panels, PMN preparation yielded populations comprised of
98–99% PMNs, as determined by CD4, CD8, CD19, CD23, CD56, and
CD64 staining (dotted lines, isotypic controls). Lower panels, PMNs were
stained with anti-CD11b, anti-CD66b, and anti-CD45 on the one hand,
with anti-CD62L, anti-CD53, and anti-CD16 on the other (dotted lines,
isotypic controls; bold lines, purified PMNs; and thin lines, PMNs in whole
PMN preparations. PMNs were obtained by Dextran T500
3998Fc?RIIIb-INDUCED PRODUCTION OF G-CSF AND GM-CSF
by guest on April 17, 2013
upper panels). The cell preparations contained ?0.1 ?g/ml endo-
toxin, and PMNs had not been activated by the isolation procedure
or hypotonic lysis, as documented by the absence of reciprocal
changes in expression of CD11b, CD66b, and CD45, which were
not up-regulated (33–35), whereas CD62L, CD53, and CD16 were
not down-regulated (36–38) in purified PMNs (Fig. 1, lower pan-
els), compared with unmanipulated PMNs in whole blood. Our
PMN preparations were extremely pure, LPS free, and nonacti-
vated, thus fulfilling the three criteria for evaluating their behavior
The proportion of spontaneously annexin V-binding PMNs de-
clined from 35.7 ? 1.5 to 16.4 ? 1.5% (p ? 0.05) after a 12-h
incubation, with 5 ?g/ml anti-CD16 F(ab?)2in six independent
experiments. Interestingly, a similar tendency was observed in the
absence of the second-layer Ab (23.2 ? 1.5% of the cells bound
annexin V, p ? 0.05, compared with 32.6 ? 2.1 in aged untreated
cells: five separate experiments). In contrast, anti-CD18 F(ab?)2
exerted no effect on spontaneous apoptosis (Fig. 2A). This finding
agrees with previous reports (39, 40). The proportion of necrotic
cells was ?5% for CD16-treated, CD18-treated, and untreated
aged PMNs, indicating that CD16 cross-linking did not induce
necrosis after a 12-h incubation (Fig. 2, inset).
For independent assessment of apoptosis, PMNs incubated with
anti-CD16 or anti-CD18 F(ab?)2were analyzed using three other
methods. Representative examples of six experiments are shown in
Fig. 3. annexin V binding is presented for comparison (Fig. 3A).
Hypoploid cells became detectable after 18 h in culture, and there
was a 37.7 ? 5.3% reduction (p ? 0.04) in CD16-treated PMNs,
compared with CD18 (Fig. 3B, histograms 3 and 4). DNA frag-
mentation in PMNs cultured alone or in the presence of anti-CD18,
but not yet in that of anti-CD16, was confirmed (Fig. 3C, lanes 2,
4, and 3). There were May-Gru ¨nwald-Giemsa-stained PMNs from
the same cultures displaying apoptotic morphology in medium
alone or following incubation with anti-CD18, then treated with
anti-CD16 (Fig. 3D, samples 2, 3, and 4).
However, it may be argued that these results could be due to the
higher expression of CD16 (ABC: 447.7 ? 1.5 ? 103, four ex-
periments), compared with CD18 (ABC: 178.4 ? 2.5 ? 103). This
seems unlikely, because the delay in apoptosis was dependent on
the amount of anti-CD16. This was detectable at a dose of 0.1
?g/ml anti-CD16, whereas, even at a dose of 10 ?g/ml, anti-CD18
did not inhibit at all annexin V binding, after a 12-h incubation
(Fig. 2B). Supporting this view is the fact that the doses required
to saturate surface CD16 and CD18 (Fig. 4) were exactly the same
(5–10 ?g/ml), even though this does not necessarily imply that
such doses produced maximal activity.
Expression of ?2integrins
The expression of adhesion molecules was evaluated in the context
of CD16-delayed apoptosis. Virtually all freshly isolated PMNs
expressed CD11b with an MFI of 7.1 ? 0.3 (mean ? SEM of five
independent experiments). After a 12-h incubation in medium, two
peaks became distinctly apparent for CD11b (Table I), in that the
MFI raised to 11.5 ? 0.7 in a 66.6 ? 2.5% fraction of the PMNs
(this CD11bbrightpopulation will be referred to as such), whereas
it declined to 2.3 ? 0.2 in the remaining 22.4 ? 1.4% fraction
(CD11dimpopulation). After a 12-h CD16 stimulation, the
CD11bbrightPMN subpopulation accounted for as few as 36 ?
2.5% of the PMNs (p ? 0.01, compared with untreated aged
PMNs), whereas 51.6 ? 1.8% of PMNs became CD11bdim(p ?
0.01, compared with untreated PMNs). A representative example
is shown in Fig. 5A. Because stimulation induces conformational
changes of CR3 and alters the accessibility of some epitopes on
CD11b/CD18, the decrease in CD11bbrightPMN may be due to the
burying of CD11b epitopes, rather than a genuine reduction in the
number of CD11b molecules. However (Table II), similar results
anti-CD16 or anti-CD18 F(ab?)2. Mouse monoclonal F(ab?)2were cross-linked with 30 ?g/ml sheep F(ab?)2anti-mouse F(ab?)2.After 12 h, the cells were
assayed for their viability and apoptotic rate using PI and Annexin VFITCstaining. A, Percentages of Annexin VFITC? cells among the PI-excluding
population were determined by FACS to evaluate the apoptotic rate (mean ? SEM of six experiments). Viability of the cells, i.e., the percentage of
PI-excluding cells, is presented in the inset (mean ? SEM of triplicate measurements). B, Dose-effect curves were conducted with increasing amounts of
anti-CD16 (filled circles) and anti-CD18 (open circles), and Annexin V-binding PMNs were enumerated after 12 h in culture (mean ? SEM of three
Anti-CD16 mAb delays the PMN apoptotic rate. Freshly isolated PMNs were cultured in medium alone, or in the presence of 5 ?g/ml
3999The Journal of Immunology
by guest on April 17, 2013
were obtained, irrespective of which of the three PE mAb (Bear 1,
44, or 2LPM19C) was used to identify CD11b.
CD16 cross-linking did not affect the expression of a number of
other PMN surface molecules, whether they are transmembrane,
such as CD15, CD32, and CD35 (Fig. 5A), or GPI linked, such as
CD48, CD55, CD59, and CD66b. Anti-CD16 treatment produced
a lowered expression of CD18, the ?-chain of CR3. This effect was
restricted to the CD11bdimsubpopulation. The same holds true for
the alternative ?-chains of the dimer, CD11a and CD11c, albeit to
a lesser degree. Representative dual fluorescence histograms are
shown in Fig. 5B, based on three independent experiments for
mAbs defining CD11b and CD18, CD11a, or CD11c. A time-
course study (Fig. 6A) revealed that the expansion in the dimmer
CD11b subpopulation correlated with the length of time the CD16
had been cross-linked by anti-Fc?RIIIb F(ab?)2. After 16 h of
CD16 stimulation, this treatment prevented 55.2 ? 2.6% (mean ?
SEM of three separate experiments) of the PMNs to become
CD11bbright. The reduction in the CD11brightpopulation was also
related to the amount of anti-CD16 F(ab?)2added to the culture
The correlation between early apoptosis and expression of
CD11b was examined by staining the cells with Annexin VFITC
and PE anti-CD11b, following a 12-h incubation with anti-CD16
or anti-CD18 F(ab?)2. In the CD16-treated PMNs, 29.5 ? 1.2% of
the CD11bdimcells underwent apoptosis, compared with 4.7 ?
0.8% of the CD11bbrightcells (p ? 0.05). Similarly, in the CD18-
treated PMNs, 63 ? 0.6% of the CD11bdimcells were apoptotic,
compared with 6.9 ? 0.6% of the CD11bbrightcells (p ? 0.04). A
representative example of six experiments is shown in Fig. 7A.
Similar to anti-CD16, which reduced apoptosis by 54.1 ? 2.3%
at a dose of 10 ?g/ml, treatment of fresh cells with 10 ?g/ml
anti-CD11b F(ab?)2decreased PCD in PMNs by 44.5 ? 2.7% after
an 18-h incubation (Fig. 8). A mixture of 5 ?g/ml anti-CD16 and
5 ?g/ml anti-CD11b showed additive effects (71.4 ? 1.5%, three
SFs are produced in response to CD16 engagement
We next investigated whether SFs were induced in response to
CD16 cross-linking. Following an 18-h incubation of 2 ? 106
sis. Apoptosis was assessed using four methods in: 1)
freshly isolated and 2) PMNs cultured in medium
alone, or in the presence of 3) anti-CD16 or 4) anti-
CD18 for 12 h (A and D) or 18 h (B and C). A, An-
nexin V binding was determined, as described in the
legend to Fig. 1. B, Hypoploidy was evaluated using
the method developed by Nicoletti et al. (see Ref. 30).
C, DNA fragmentation was confirmed by electro-
phoresis on 1% agarose gel. D, Apoptotic morphol-
ogy was detected (arrows) on air-dried slides by May-
Gru ¨nwald-Giemsa staining of the cells (original
Confirmation of the delay in apopto-
of 105freshly isolated PMNs was incubated with increasing amounts of
unconjugated anti-CD16 (filled circles), anti-CD18 (open circles), or anti-
HLA-I (filled squares) F(ab?)2, and the binding developed with FITC-la-
beled sheep F(ab?)2anti-mouse IgG F(ab?)2, which served as a negative
control (open squares). The MFI of the second-layer reagents alone were
systematically substracted from those obtained with previous incubation
with anti-CD16, anti-CD18, or anti-HLA-I F(ab?)2. Data represent the
mean ? SEM of three experiments.
Saturation of the membrane Ags by the related Abs. A total
Table I. CD11bbrightand CD11bdimsubpopulations of untreated aged
and CD16-treated aged PMNsa
66.6 ? 2.5
11.5 ? 0.7
36.0 ? 2.5
9.5 ? 0.3
p ? 0.01
22.4 ? 1.4c
2.3 ? 0.2
51.6 ? 1.8
2.6 ? 0.2
p ? 0.01
aThe expression of CD11b was evaluated after a 12-h incubation in medium or
following a 12-h treatment of PMNs with anti-CD16 F(ab?)2.
bMann-Whitney U test between unstimulated aged PMNs and CD16-treated
cMean ? SEM of five independent experiments.
4000Fc?RIIIb-INDUCED PRODUCTION OF G-CSF AND GM-CSF
by guest on April 17, 2013
PMNs in medium or in the presence of increasing amounts of
F(ab?)2specific for CD16, CD18, or HLA-I, the PMN-conditioned
supernatants of these primary cultures were collected and used as
the culture medium for 5 ? 105freshly explanted PMNs. CD18
mAb was taken as a negative control, and, because ligation of
HLA-I molecules retards somewhat apoptosis (47), anti-HLA-I
mAb served as a positive control. Annexin V-binding fresh PMNs
were enumerated after another 18 h in culture, and the supernatant-
induced reduction of apoptosis was calculated. These PMNs were
rescued from spontaneous apoptosis by CD16-treated PMN super-
natants in a dose-dependent manner, whereas, as expected, there
was a 25% inhibition in cells treated with anti-HLA-I mAb, and
CD18-conditioned supernatants produced no effect on apoptosis of
fresh PMNs (Fig. 9A). Furthermore, PMN-PMN interactions do
not seem to be involved in the production of SFs. To rule out this
possibility, PMNs were cultured at a variety of different cell den-
sities in medium alone or in the presence of 5 ?g/ml anti-CD16 or
anti-CD18. As above, the supernatants were added to a constant
number of fresh PMNs (0.5 ? 106), which were examined 18 h
later (Fig. 9B). The PCD-inhibiting effect of the supernatants was
the same, as indicated by similar percentages of Annexin V-bind-
ing cells, irrespective of the number of PMNs present in the pri-
Role of G-CSF and GM-CSF
G-CSF and GM-CSF emerged as credible agents in the CD16-
mediated reduction of apoptosis (6, 17, and our pilot experiments).
Increasing amounts of G-CSF, GM-CSF, and half G-CSF plus half
GM-CSF were added to PMNs without CD16 engagement (Fig.
10). The antiapoptotic effect was calculated after 18 h in culture,
compared with PMNs in medium alone. At a dose of 10 ng/ml,
G-CSF, GM-CSF, and G-CSF plus GM-CSF reduced Annexin V
binding by 62.5 ? 0.9, 62.7 ? 0.1, and 86.2 ? 0.4%, respectively
(mean ? SEM of three experiments).
RT-PCR experiments were performed to test this hypothesis.
M? and B cells served as positive controls for IL-6 and CD23
transcripts, and provided additional evidence (Fig. 10A) for the
lack of contaminating M? and eosinophils in the preparations of
fresh PMNs, in which the message for CD23 and IL-6 was unde-
tectable. In contrast, the former was expressed in B cells, and the
latter in resting M?, IL-1?-activated M?, and B cells. As shown
in Fig. 11B, when cultured in the presence of anti-CD16 F(ab?)2,
transcription of mRNA for G-CSF was evident by 3 h (G-CSF/?-
actin: 30), reached a maximum at 6 h (G-CSF/?-actin: 59.6), and
declined at 12 h (G-CSF/?-actin: 5.1). It was also found that, to a
lesser degree, mRNA for GM-CSF peaked at 6 h (GM-CSF/?-
actin: 24.2), and plateaued until at least 12 h.
We next determined whether, following CD16 cross-linking, the
transcription of mRNA for G-CSF and GM-CSF resulted in pro-
tein synthesis (Table III). Substantial quantities of G-CSF, and
lower quantities of GM-CSF were detected in the PMN superna-
tant. The levels of both cytokines paralleled the amounts of CD16
mAb used to stimulate the cells.
A prerequisite was to verify that anti-G-CSF and anti-GM-CSF
mAbs were strictly specific. After SDS-PAGE, recombinant hu-
man G-CSF (5 ?g/ml) and GM-CSF (1 ?g/ml) were transferred
onto polyvinylidene difluoride membrane and probed with both
duces down-regulation of ?2integrins.
A, CD11b, and CD35 expression on
PMNs was analyzed before and after a
12-h incubation in medium or with 5
?g/ml anti-CD16 F(ab?)2. Percentages
and MFIs of each population were ob-
tained by FACS using FITC anti-CD11b
and FITC anti-CD35 (as a negative con-
trol) mAbs. Data are representative of
five separate experiments. B, Dual fluo-
isolated (left panel) or stimulated for 12 h
with 5 ?g/ml anti-CD16 F(ab?)2(right
CD11b and PE anti-CD18, anti-CD11a, or
anti-CD11c mAbs. The same results were
obtained in two other experiments.
CD16 cross-linking in-
Table II. Distribution of CD11bbrightand CD11bdimPMN populations
following CD16 stimulation using three different anti-CD11b mAbsa
Percentage of CD11b Positive Cells (MFI)
Incubated with anti-CD18
Incubated with anti-CD16
aPMNs, isolated by Ficoll centrifugation, were cultured with 5 ?g/ml control
(anti-CD18) or anti-CD16 F(ab?)2for 12 h. CD11b expression was then analyzed
using three different PE-conjugated anti-CD11b mAbs (Bear 1, 44, and 2LPM19c).
Percentage and MFI were determined by FACS. Data are representative of two sep-
4001The Journal of Immunology
by guest on April 17, 2013
Abs. Neither of these Abs exhibited cross-reactivity (data not
shown). To confirm the functional activity of G-CSF and GM-CSF
released by CD16-stimulated PMNs in primary culture, fresh
PMNs were incubated SF(s)-containing supernatants in the ab-
sence or in the presence of neutralizing Abs. Annexin V binding
was measured 18 h later, and variations were calculated by com-
parison with PMNs purified from the same samples and cultured
without Ab for the same period of time. As shown in Fig. 12,
CD16-conditioned supernatants diminished the percentages of an-
nexin V-positive cells by 52.7 ? 1.9% (mean ? SEM of five
separate experiments). Although anti-TNF-? Ab did not influence
the antiapoptotic effect of SFs (still reduction of apoptosis was
52.4 ? 1.7%), anti-G-CSF and anti-GM-CSF generated 31.5 ? 2.7
and 33.1 ? 1% abrogation of the antiapoptotic effect produced by
the primary culture supernatants (p ? 0.04, compared with CD16-
conditioned supernatant alone). The capacity of SFs to inhibit ap-
optosis was almost completely blocked (5.9 ? 1.4% reduction,
p ? 0.04) with a mixture of anti-G-CSF and anti-GM-CSF Abs,
suggesting that these two cytokines produced an additive effect,
and accounted for most of the protection induced by treatment of
PMNs with anti-CD16.
Bax expression in PMNs treated with conditioned supernatant
Additional experiments were performed to refine the understand-
ing of the mechanism that underlies the antiapoptotic effect of
G-CSF and GM-CSF. Direct evidence has been provided recently
(42) for a key role of Bax as a proapoptotic molecule in PMNs.
This finding was applied to our model. PMN membrane staining of
CD11b, coupled with intracellular staining of Bax, was performed
in PMNs cultured for 18 h either in primary nonconditioned or in
CD16-stimulated supernatants. After another 18 h in culture, the
?g/ml anti-CD16 F(ab?)2for various periods of time. PMNs were then stained with FITC anti-CD11b mAb (Bear 1), and percentages of CD11bdim(open
symbols) and CD11bbrightcells (closed symbols) were evaluated by FACS. B, PMNs were incubated for 12 h with different concentrations of anti-CD16
F(ab?)2. Percentages of CD11bdimcells were enumerated by FACS. Data represent the mean and SEM of three experiments.
CD16-induced reduction of CD11b expression is time and dose dependent. A, PMNs were incubated with (circles) or without (triangles) 5
hand, and Annexin V binding and Bax expression on the other. A, CD16-
or CD18-treated PMNs were double stained using Annexin VFITCand PE
anti-CD11b, and the percentages of annexin V?in the CD11bbrightand the
CD11dimcells were enumerated (six experiments). B, CD16- or CD18-
treated PMNs were labeled with PE anti-CD11b mAb, washed three times,
permeabilized in 1 ml 0.1% saponin, incubated with rabbit anti-Bax
F(ab?)2, washed another three times, and developed with FITC goat F(ab?)2
anti-rabbit F(ab?)2. The MFIs of Bax in the CD11bbrightand the CD11bdim
cells were recorded (three experiments).
Correlation between membrane CD11b density on the one
anti-CD11b, or anti-CD16 plus anti-CD11b. As described in the legend to
Fig. 2, fresh PMNs were cultured in medium alone, or in the presence of
anti-CD16 F(ab?)2, anti-CD11b F(ab?)2, or anti-CD16 F(ab?)2plus anti-
CD11b F(ab?)2for 12 h (mean ? SEM of three experiments).
Reduction of PMN apoptosis by cross-linking anti-CD16,
4002Fc?RIIIb-INDUCED PRODUCTION OF G-CSF AND GM-CSF
by guest on April 17, 2013
percentage and MFI of Bax-containing cells were down-regulated
(Table IV and Fig. 7B) by CD16 cross-linking in PMNs. Interest-
ingly, although significant also in the CD11bdimpopulation, this
reduction predominated in the CD11bbrightpopulation.
Effect of CD16 on caspase-3
The involvement of caspase-3 in spontaneous apoptosis of PMNs
(9) prompted us to determine the effect of this protease, following
incubation of the cells with anti-CD16 F(ab?)2. Although it was
inactive in untreated fresh cells (Fig. 13), aged PMNs developed a
strong caspase-3 activity, which was retained in the presence of
anti-CD18 F(ab?)2used as a negative control. This was completely
abolished by Ac-DEVD-CHO, an inhibitor for caspase-3, demon-
strating the specificity of the used assay. Incubation of PMNs in
the presence of anti-Fc?RIIIb F(ab?)2resulted in a 26 ? 2.7%
reduction (mean ? SEM of four separate experiments) of caspase-3
activity (p ? 0.05, compared with anti-CD18).
The central message from this report is that Fc?RIIIb long-term
cross-linking generates the production of G-CSF and GM-CSF,
and thereby retards spontaneous apoptosis of senescent PMNs. Ob-
viously, it cannot be inferred from this finding that other apoptotic
processes of PMNs, such as Fas-mediated apoptosis, steroid stim-
ulation, or radiation exposure, would also be affected by CD16
stimulation. After 18 h in culture, the cell population has not yet
started to exhibit significant evidence of necrosis, as previously
highlighted by Savill et al. (1). In this study, it was essential to rule
out that contaminating LPS, an apoptosis-delaying stimulus (6, 28)
possibly present in the reagents, was responsible for the PMN sur-
vival or worked in synergy with CD16 ligation to cause the effect.
In this study, F(ab?)2of the CD16 mAb 3G8 were incubated
with the cells and cross-linked with a second Ab. Furthermore,
albeit to a much lower extent, the same tendency was observed in
the absence of the second-layer Ab. Thus, in contrast to the 10-min
culture, in which an extensive cross-linking of surface Fc?RIIIb is
required to transduce signals (43), pairing of these molecules
seems to be sufficient in an 18-h culture to trigger the earliest
events of transduction. Such a phenomenon might perhaps be as-
signed to the extreme abundance of Fc?RIIIb molecules on the
PMN surface (Ref. 44, and this study). Our finding, which is not
this surprising, is consistent with the demonstration that Fc?RIIIb
cross-linking is not an absolute requirement for signaling functions
of PMNs (45).
F(ab?)2to CD16 (filled circles), to HLA-I (filled squares), or to CD18 (open circles). These primary culture supernatants were collected and added to 0.5 ?
106fresh PMNs, while control PMNs were left in medium. Following another 18-h incubation, annexin V?/PI?PMN were enumerated in these secondary
cultures. B, PMNs were incubated at various densities (0.5–5 ? 106/ml) in medium alone (open squares) or in the presence of 5 ?g/ml anti-CD16 (filled
circles) or anti-CD18 (open circles) F(ab?)2. The supernatants were added to 0.5 ? 106fresh PMNs, and these cells were examined for annexin V binding
18 h later. Supernatant-induced variations in apoptosis were calculated according to the formula: (control apoptotic PMNs ? supernatant apoptotic
PMNs/control apoptotic PMNs) ? 100. The results are means ? SEM of triplicate experiments.
SFs are produced by PMNs in response to CD16 engagement. A, PMNs (2 ? 106) were incubated for 18 h with increasing amounts of mAb
(lane 3), CD23 (lane 4), and ?-actin (lane 5). Resting monocytes (M?) and activated monocytes (IL-1?, 0.5 ng/ml for 4 h) served as positive control for
IL-6, G-CSF, and GM-CSF. B cells served as positive control for CD23. Data are representative of two experiments. ND, Not done. B, Induction of G-CSF
and GM-CSF mRNA expression by mouse anti-F(ab?)2anti-CD16 at concentration of 10 ?g/ml in a time-dependent manner from 0 to 12 h. Data are
representative of two experiments.
RT-PCR analysis. A, mRNA expression obtained by RT-PCR and agarose gel electrophoresis for G-CSF (lane 1), GM-CSF (lane 2), IL-6
4003The Journal of Immunology
by guest on April 17, 2013
Given the homeostatic role attributed to the ?2integrins in the
programmed elimination of PMNs (39, 46), the effect of CD16
activation on the expression of CD11b/CD18 was then investi-
gated. Spontaneously apoptotic PMNs have been shown to express
CD11b/CD18 and CD11c/CD18 at increased levels (46), and a
20-min cross-linking of CD16 demonstrated to promote this en-
hancement (47). We found that, following this initial up-regulation
of several ?2integrins secondary to a rapid translocation of the ?-
and the ?-chains from the intracellular pool to the cell surface (33),
CD16 induced the persistence of significantly less CD11bbright
PMNs in 12-h cultured cells, compared with PMNs maintained in
medium. Conformational changes of CD11b caused by CD16
long-term engagement and masking certain epitopes are unlikely
to account for this reduction, because three different anti-CD11b
mAbs produced similar results. However, it is interesting to note
that the MFI of the dimmer CD11b subpopulation was 10-fold
higher with the 44 than the Bear 1 mAb, suggesting that the I
domain epitope recognized by 44 and the 2LPM19c mAbs re-
mained more accessible than the Bear 1 epitope during CD16 ac-
tivation (48). Nonetheless, treatment of PMNs with anti-CD11b
F(ab?)2delayed apoptosis of aging cells. This finding confirms and
extends our previous report that soluble Fc?RIIIb prolongs the
survival of PMNs in vitro (16), presumably through CRs (49). The
effects of anti-CD16 and anti-CD11b were additive. Ligation of
CD16 may favor the binding of its polysaccharides to the lectin
site of CD11b (50), whereas the anti-CD11b mAb Bear 1 used in
our experiments is specific for this part of CD11b (51). Such data
are consistent with the conclusion that CD16 polysaccharides and
Bear 1 share overlapping, but not identical binding sites on the
CD11b lectin site. Our finding that the anti-CD11b, along with the
anti-CD18 mAb treatment, exerted additive effects on PMN sur-
vival is consistent with, but does not prove, the model of functional
interactions between these receptors. Furthermore, the fact that
anti-CD18 had no effect on survival argues against such interac-
tions. However, similar results have been reported by Watson et al.
(40). Using the mAbs that we used in our own study, these inves-
tigators showed that cross-linking of the ?-chain of the ?2inte-
grins, CD11a and CD11b, significantly delayed the apoptosis of
resting PMNs, whereas cross-linking of the common ?-chain,
CD18, failed to alter the apoptotic rate of the cells. In theory,
CD16 might however cooperate with CD11b in apoptosis-delaying
signal transduction. In addition, although C is not involved,
CD11b/CD18 and CD16 cooperate in adhesiveness, phagocytosis,
and the generation of respiratory burst (39, 52–54). As a result, the
CD16-induced augmentation in the proportion of CD11bdim
should generate a functional alteration of PMNs, and this was ac-
tually the case (Ref. 15 and data not presented).
There appeared that the CD11bdimpopulation became apoptotic,
as reported by Jones and Morgan (55). It follows that long-term
engagement of CD16 seems to put more cells at risk of becoming
apoptotic. In support of this view is the demonstration that in-
creased PMN accumulation in CD11b/CD18-null mice in thiogly-
colate-induced peritonitis is accompanied by decreased apoptosis
of extravasated PMNs (45). Interestingly, although predominating
in the CD11bbrightpopulation, there was a down-regulation of Bax
in the CD11bdimpopulation. CR3 does not initiate apoptosis by
itself, but potentiates TNF-?-mediated (39), and possibly sponta-
neous apoptosis of PMNs. We failed to establish the involvement
of PMN-PMN interactions in the prolonged survival, inasmuch as
the conditioned supernatants of increasing numbers of cells pro-
duced almost the same PCD-inhibiting effect when incubated with
a constant number of PMNs for a further 18 h.
G-CSF and GM-CSF are present at sites of neutrophilic inflam-
mation. In this respect, our major finding was that PMNs release
substantial quantities of cytokines in response to CD16 cross-link-
ing, explaining in part the delayed apoptotic process (56). It is well
established that, among other agents, these growth factors exert a
potent effect on the regulation of apoptosis in PMNs (57, 58). The
physiologic control of G-CSF and GM-CSF production remains
only partially understood. G-CSF (59) and GM-CSF (60) are pro-
duced by a variety of cell types, following appropriate stimulation;
in this study, we provide some unanticipated evidence for the
CD16-initiated transcription of their mRNAs in PMNs. This is not
constitutive (17), as confirmed by the absence of transcripts in
ptosis of PMNs. A total of 0.01–10 ng/ml G-CSF, GM-CSF, or 50% G-
CSF plus 50% GM-CSF was added to 0.5 ? 106fresh PMNs, and their
antiapoptotic effect was evaluated after an 18-h incubation. Variations in
annexin V binding were calculated as in the legend to Fig. 9.
G-CSF, GM-CSF, and G-CSF plus GM-CSF delay apo-
Table III. PMN produce G-CSF and GM-CSF in response to CD16 engagement, but not CD18 engagement, in a dose-dependent mannera
mAb F(ab?)2Used to
G-CSF (pg/ml) GM-CSF (pg/ml)
Expt. 1Expt. 2 Expt. 3Expt. 1Expt. 2Expt. 3
aThe production of G-CSF and GM-CSF was measured following a 18-h incubation of 5 ? 105PMNs with 0–20 ?g/ml mAb F(ab?)2to CD16 or CD18.
4004 Fc?RIIIb-INDUCED PRODUCTION OF G-CSF AND GM-CSF
by guest on April 17, 2013
fresh PMNs. The proteins became detectable after 18 h of culture
in the presence of cross-linked anti-CD16 F(ab?)2. To ensure that
the proteins measured in the culture supernatants were not released
by contaminating M?, it was essential to yield pure populations of
PMNs. This concern was clearly addressed, because our cell sus-
pension was comprised of 99% PMNs. The absence of contami-
nating M? and eosinophils was confirmed by the fact that mRNA
for IL-6 and CD23 were undetectable in fresh PMNs. Neutraliza-
tion of G-CSF biologic activity by specific antiserum abrogated
G-CSF-mediated inhibition of PCD. In addition, immunodepleting
GM-CSF blocked the effect of GM-CSF. These data suggest that
G-CSF and GM-CSF produce distinct, but additive antiapoptotic
effects. Comparable results have been reported by Matute-Bello et
al. (61) in bronchoalveolar lavage fluid from patients with the
acute respiratory distress syndrome. It is known that GM-CSF can
induce the secretion of G-CSF by PMNs (62). It would thus be
interesting to study the kinetics of GM-CSF and G-CSF production
in the CD16-stimulated PMN model, to address the issue as to
whether the G-CSF expression results from that of GM-CSF. It is
surprising that 5 ?g/ml F(ab?)2anti-CD16 was sufficient for max-
imal activity of conditioned supernatants (see Fig. 6), although
doses as high as 20 ?g/ml still induced the production of larger
amounts of G-CSF and GM-CSF (see Table III). As previously
described (63), this discrepancy suggests that subsaturating dos-
ages of mAb bind uniformly to all the cells, but a mAb dose-
dependent proportion of those mAb-bound PMNs responded with
a release of G-CSF and GM-CSF.
The precise mechanism by which GM-CSF and G-CSF extend
the survival of PMNs warrants further analysis. To begin to ad-
dress this issue, the level of the death promoter Bax was measured
and found to be reduced by CD16 long-term engagement. This is
consistent with the recent report that, in neutrophilic inflammatory
diseases, the delay in PMN apoptosis was associated with mark-
edly decreased levels of Bax, and normalized by stimulation with
G-CSF and GM-CSF in vitro (45).
Caspase-3 has been reported to be pivotal in spontaneous apoptosis
of PMNs (9). In the present study, CD16-stimulated PMNs reduced,
but did not abolish activity of this cysteine protease. Such a finding is
consistent with the observation that impaired apoptotic death signal-
ing in inflammatory lung PMNs is associated with decreased, but not
totally inhibited expression of caspases (40). One possibility is that,
due to insufficient activation by caspase-3, protein kinase C? does not
acquire the capacity to stimulate phospholipid scramblase (64), and to
induce the ensuing translocation of phosphatidylserine to the outer
leaflet of the plasma membrane. There appear to be at least two
explanations. Caspase-3 might be one of several different molecular
of no surprise that the CD16-induced inhibition of apoptosis remains
partial. It has nevertheless been established that PCD may occur
through apparently caspase-3-independent ways (65). Although it has
not been clearly defined in humans, PML nuclear bodies, which are
nuclear matrix-associated structures inducing a caspase-independent
death process (66), might follow another pathway in PMN spontane-
ous apoptosis. Alternatively, this spontaneous apoptosis might be
exclusively mediated by caspase-3. In the latter scenario, it is possible
that caspase-3 becomes accessible to CD16-induced signals in only a
fraction of the cells, presumably depending on their age. Indeed,
terminally differentiated PMNs undergo major functional changes as
population of cells. Relatively low levels of reactive oxygen species
generated in this model (68) do not prevent caspases from function-
ing, and offer another explanation for the incomplete inhibition of
caspase-3 following CD16 engagement.
CD16-stimulated PMN. The experiment was conducted as described in the
legend to Fig. 9, except that the primary culture supernatants were prein-
cubated with anti-TNF-?, anti-GM-CSF, anti-G-CSF, or anti-GM-CSF
plus anti-G-CSF Abs for 90 min at 37°C, before being added to the fresh
PMNs. The results are means ? SEM of triplicate experiments.
Prevention of the survival effect of factors released by
Freshly isolated PMNs untreated, or stimulated for 12 h with 5 ?g/ml
anti-HLA-I or anti-CD16 F(ab?)2cross-linked with 30 ?g/ml sheep F(ab?)2
anti-mouse F(ab?)2, were lysed. Cell lysates were analyzed for caspase-3
activity using a colorimetric assay. As a control, cell lysate PMNs from
anti-HLA-I-stimulated PMN were analyzed for caspase-3 activity in the
presence of Ac-DEVD-CHO, a caspase-3 inhibitor. Data are mean ? SEM
of three separate experiments.
CD16 engagement reduces the activity of caspase-3.
Table IV. Supernatants of CD16-stimulated PMNs in primary cultures
reduce the level of Bax in fresh PMNs after another 18 h in secondary
Intracellular Bax in Secondary PMNs
Cultured in CD16-
% positive cells
% positive cells
% positive cells
37.5 ? 1.2b
7.55 ? 0.09
7.2 ? 0.3
7.13 ? 0.55
12.1 ? 0.8
5.62 ? 0.28
1.3 ? 0.5
3.86 ? 0.11
99.1 ? 0.4
8.45 ? 0.27
93.7 ? 1.4
7.75 ? 0.58
aPMNs were cultured with or without CD16 F(ab?)2for 18 h (primary cultures),
and fresh PMNs were incubated for another 18 h in the primary culture supernatants.
PMN membrane staining of CD11b was coupled with intracellular staining of Bax in
PMNs collected from secondary cultures.
bMean ? SEM (five experiments).
4005The Journal of Immunology
by guest on April 17, 2013
In conclusion, CD16 long-term stimulation significantly pro-
longed the survival of PMN. This phenomenon was associated
with induction of mRNA and protein synthesis of G-CSF and GM-
CSF. PMN-binding anti-Fc?RIIIb autoantibodies might produce
the same effect, accompanied by the absence of up-regulation of ?2
integrins. CD16 and the related autoantibodies may thus be influ-
ential in the control of inflammatory responses, and thereby facil-
itate the development of autoimmune states.
We acknowledge Roger Casburn-Budd (The Binding Site, Birmingham,
U.K.) for critical reading of the manuscript. We are indebted to Guillaume
Le Bigot (Beckman Coulter France) for the generous gift of reagents.
Thanks are also due to Simone Forest and Sylvie Hamon for expert sec-
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