Polymorphonuclear Leukocytes Modulate
Tissue Factor Production by Mononuclear
Cells: Role of Reactive Oxygen Species
This information is current as
of July 31, 2017.
Yves Cadroy, Dominique Dupouy, Bernard Boneu and Henri
Plaisancié
J Immunol 2000; 164:3822-3828; ;
doi: 10.4049/jimmunol.164.7.3822
http://www.jimmunol.org/content/164/7/3822
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References
Polymorphonuclear Leukocytes Modulate Tissue Factor
Production by Mononuclear Cells: Role of Reactive Oxygen
Species
Yves Cadroy,1* Dominique Dupouy,* Bernard Boneu,* and Henri Plaisancié†
T
hrombosis and atherosclerosis are multicellular processes
in which the role of leukocytes is becoming increasingly
recognized. Monocytes/macrophages express procoagulant activities (PCA)2 which are mediated to a large extent by cell
surface-associated tissue factor (TF) (1– 4). TF is the cellular receptor and cofactor for plasma factor VII(a), which initiates the
coagulation protease cascade leading ultimately to the generation
of thrombin and fibrin and is the main initiator of thrombogenesis
in vivo (5). Quiescent monocytes synthesize and express TF on
their membranes in reponse to a wide variety of stimulating agents,
including LPS, cytokines, arachidonic acid, and immune complexes (6, 7).
Polymorphonuclear leukocytes (PMN) are another type of leukocyte whose role in several pathological processes related to
thrombosis and atherosclerosis appears important (8). They act notably by releasing potent mediators which may interact with other
blood cells. These mediators include proteolytic enzymes such as
cathepsin G, and reactive oxygen species (ROS). Cathepsin G is a
platelet-activating agent (9 –11). ROS are derived from molecular
oxygen by sequential monovalent reductions, yielding the superoxide radical (O2⫺), hydrogen peroxide (H2O2), and the hydroxyl
radical (䡠OH). A number of recent studies have shown that their
*Laboratoire de Recherche sur l’Hémostase et la Thrombose, and †Isoprim, Toulouse,
France
Received for publication October 29, 1999. Accepted for publication January
21, 2000.
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.
1
Address correspondence and reprint requests to Dr. Yves Cadroy, Laboratoire de
Recherche sur l’Hémostase et la Thrombose, Pavillon Lefèbvre, CHU Purpan, 31059
Toulouse Cedex, France. E-mail address: [email protected]
2
Abbreviations used in this paper: PCA, procoagulant activity; TF, tissue factor;
PMN, polymorphonuclear leukocytes; ROS, reactive oxygen species; NAC, N-acetyl
cysteine; X, xanthine; XO, xanthine-oxidase; PDTC, pyrrolidine dithiocarbamate;
LDH, lactate dehydrogenase; AU, arbitrary units; DR, HLA-DR class II MHC Ag.
Copyright © 2000 by The American Association of Immunologists
targets include endothelial cells, vascular smooth muscle cells, and
macrophages and that increased or uncontrolled ROS production is
involved in the formation of thrombosis and atherosclerosis (12).
For example, they act on endothelial cells and alter their production of prostacyclin and nitric oxide, two molecules with important
endogenous antiplatelet and vasodilatator properties (8). They induce the expression of TF (13) and increase the adhesiveness of
endothelial cells for PMN (14).
In the present study, we describe a new mechanism by which
PMN may play an important role in the pathogenesis of thrombosis
and atherosclerosis. We show that they modulate the TF production of PBMC, and that, depending on the experimental setting,
this modulation is positive or negative. It appears to be mediated
by the PMN production of ROS, underlining the role of these
mediators in the development of these pathological processes.
Materials and Methods
Materials
PBS (pH 7.4) was obtained from Seromed, Biochrom (Berlin, Germany).
Ficoll-Hypaque PLUS was purchased from Pharmacia Biotech (Uppsala,
Sweden). M199 was purchased from ATGC Biotechnologie (Noisy-leGrand, France). All other reagents were obtained from Sigma (Saint Quentin Fallavier, France).
fMLP was dissolved in 100% DMSO. N-acetyl cysteine (NAC) was
dissolved at 200 mmol/L in deionized water and neutralized by titration
with NaOH. Xanthine (X) was dissolved in NaOH. All other reagents were
dissolved in deionized water. In experiments involving pharmacological
reagents, control experiments were always performed with the corresponding solvents.
Cell isolation and cell cultures
PMN and PBMC were isolated from healthy volunteers (12 females and six
males, aged 20 –50 years) that had not taken aspirin or other nonsteroidal
anti-inflammatory drugs in the 7 days preceding the donation. Whole blood
was obtained with a 19-guage needle, anticoagulated with trisodium citrate
(0.129 M; Becton Dickinson, Meylan, France), and centrifuged at 280 ⫻ g
for 15 min at 4°C. Platelet-rich plasma was removed. The sedimented cells
were diluted to twice the original blood volume with PBS and layered onto
0022-1767/00/$02.00
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To determine whether polymorphonuclear leukocytes (PMN) modulate the production of tissue factor (TF) by monocytes, PBMC
were incubated with increasing concentrations of PMN. PMN did not express any procoagulant activity. After 20-h cocultures,
PMN enhanced or inhibited the TF production of PBMC, and this effect depended on the PMN/PBMC ratio. When the ratio
increased from 1/1000 to 1/5, without or with LPS, the TF activity of PBMC increased to peak at 2.5-fold the baseline value (p <
0.01). The TF Ag and TF mRNA also increased. This potentiating effect was mediated by reactive oxygen species (ROS) released
by PMN during the coculture; it did not require direct cell contact between PMN and PBMC, it was enhanced when PMN were
stimulated by fMLP (a chemotactic peptide), and it was inhibited by two antioxidants, N-acetyl cysteine and pyrrolidine dithiocarbamate. In contrast, when the PMN/PBMC ratio was further increased from 1/2 to 2/1, the PBMC TF activity, Ag, and mRNA
decreased and were inhibited compared with those of PBMC cultured alone (p < 0.01). This inhibitory effect required direct cell
contact between PMN and PBMC, and it was not due to a PMN-mediated cytotoxicity. To confirm the role of ROS, H2O2 enhanced
then inhibited the TF activity of PBMC in a dose-dependent manner, similarly to PMN. Thus, PMN may play an important role
in the pathogenesis of thrombosis and atherosclerosis by exerting concentration-dependent regulatory effects on the TF production
by PBMC via the release of ROS. The Journal of Immunology, 2000, 164: 3822–3828.
The Journal of Immunology
Measurement of PCA
PCA was measured on intact cells using a one-step plasma recalcification
time assay. After incubation, plates were centrifuged for 10 min at 400 ⫻
g. The supernatant was collected, and cells or supernatant (80 ␮L) were
incubated for 2 min at 37°C with citrated normal human platelet-poor
plasma (100 ␮L). In selected experiments, the PCA was measured using a
factor VII-deficient plasma (Stago, Asnières, France). Then, 100 ␮L of 25
mmol/L CaCl2 was added to initiate the reaction. The change in optical
density at 405 nm was quantitated using a microplate reader. Coagulation
times were converted into arbitrary units (AU) using reference curves determined with a standard human brain TF preparation containing 106
AU/ml (Thromborel Behring, Marburg, Germany); the logarithm of the
PCA was related to the logarithm of the coagulation time. The PCA was
expressed in AU/106 PBMC. The PCA of PBMC cells was characterized
by incubating the cells with a mixture of two mouse anti-human TF mAbs
(10 ␮g/ml; American Diagnostica, Greenwich, CT) for 30 min at 37°C.
Measurement of TF Ag
TF Ag was measured on cell lysates by commercially available immunoenzymoassay (Imubind Tissue Factor, American Diagnostica). Cell lysates were prepared by lysing PBMC in PBS containing 0.1% Triton
X-100, 1 mmol/L EDTA, 16 mmol/L octyl phosphated Dulbecco’s glucopyranoside, 10 ␮mol/L pepstatin A, 10 ␮mol/L leupeptine, 0.1 mmol/L
PMSF, and 100 Kallicrein International Units/ml aprotinin. The cell lysates
were then frozen and thawed three times. They were stored at ⫺80°C until
assayed.
TF RT-PCR
Semiquantitative RT-PCR was used to calculate relative changes in TF
mRNA levels. To correct variations in amplification efficiency in each
reaction, both TF and HLA-DR class II MHC Ag (DR) mRNAs were
detected. We chose DR as a standard because, like TF, DR is a receptor
expressed in the monocytes but not in PMN (15). Total cellular RNA was
extracted from leukocytes with a commercial kit (RNeasy Blood Mini Kit,
Qiagen, Courtaboeuf, France), and the yields per vial were 2–3 ␮g, which
is close to the expected theoretical yield. Total RNA was reverse transcribed using a commercial kit (Ready-to-go RT-PCR, Pharmacia Biotech)
for 30 min at 42°C with oligo(dT) (Pd(T)12–18) according to the manufacturer’s instructions. After inactivation of reverse transcriptase for 3 min at
94°C and addition of primers, PCR reactions were run in a Hybaid thermal
cycler (PCR express; Teddington, U.K.). The reaction mixtures were amplified for 22 cycles and the reaction cycles were 94°C for 30 s, 58°C for
20 s, and 72°C for 20 s, with final elongation at 72°C for 7 min. Sense and
antisense primers for TF were those described by Bartha et al. (16). The
forward DR primer was 5⬘-GTA AGG CAC ATG GAG/GTG ATG G-3⬘
(DNA nos. 2529 –2543 and 3282–3288), and the reverse DR primer was
5⬘-GGA CAG ATA ACG GAA AAG GAC-3⬘ (DNA nos. 3469 –3489).
All primers were labeled with Texas Red, a fluorescent probe. Amplification products were subjected to electrophoresis in 4% polyacrylamide gels.
The relative levels of TF mRNA were quantified by densitometric scanning
using an imaging densitometer (Vistra DNA sequencer 725, Amersham
International, Amersham, U.K.) and normalized according to the level of
DR mRNA. There were no detectable DR transcripts in PMN, and the level
of DR transcripts did not change when PBMC were treated with PMN.
Measurement of superoxide anion generation
Superoxide anion generation by PMN was measured as the superoxide
dismutase-inhibitable reduction of ferricytochrome-c. Increasing numbers
of PMN (0 –5 ⫻ 106 cells/ml) were incubated in 96-well microtiter plates
with cytochrome-c (75 ␮mol/L) and D-glucose (7.5 mmol/L) in M199. In
selected experiments, the cells were also incubated with LPS (100 ng/ml)
and/or fMLP (106 ␮mol/L). Immediately after addition of PMN, or fMLP
when this agonist was used, the absorbance of the reaction wells was measured at 550 nm in a microplate spectrophotometer. The readings were
repeated for 80 min. Each reaction was performed against an identical
control reaction, which contained 300 U/ml superoxide dismutase. Production of superoxide anion was determined by use of the molar extinction
coefficient of cytochrome-c (6.3 with a light path of 2 mm).
Statistical analysis
All results are expressed as mean ⫾ 1 SEM. In comparisons of two groups,
probability values were calculated by Student’s t test. In experiments involving comparisons of multiple groups, the probability that differences
existed between the means of the groups was determined by ANOVA, and
then by a Neuman-Keuls test when the p value was ⱕ0.05. Differences
were considered to be statistically significant when p was ⱕ0.05.
Results
Effect of PMN on the PCA of PBMC
After 20 h of culture, isolated PMN did not express any significant
PCA, whereas isolated resting PBMC expressed a low but detectable level of PCA (Fig. 1). When PBMC were incubated with
increasing concentrations of PMN for 20 h, the PCA expressed by
cocultured cells (Fig. 1) or present in the supernatant (data not
shown) was modified depending on the number of PMN added to
PBMC. The PCA present in the supernatant constituted approximately one-third of the total PCA and was probably related to the
presence of cell-derived microparticles (17).
When the PMN/PBMC ratio increased from 1/1000 to 1/5, the
cell PCA significantly increased to peak at 2.5-fold the baseline
value ( p ⬍ 0.01). This effect was not due to a contamination of
PMN by endotoxin because the effect was comparable when PMN
were incubated with PBMC in the presence of polymyxin B (10
␮g/ml).
When the PMN/PBMC ratio was further increased to 2/1, the
cell PCA decreased and, at the highest investigated PMN/PBMC
ratio, it was significantly inhibited compared with that of isolated
PBMC ( p ⬍ 0.01). This effect was not due to a PMN-mediated
cytotoxicity of PBMC, as shown by the measurement of LDH, a
cytoplasmic enzyme released by damaged cells, in the cell culture
supernatant. During the 20-h culture, 2.5 ⫻ 106 PBMC alone released 0.98 ⫾ 0.32 mU/ml of LDH (n ⫽ 5). Because the total
amount of LDH present in 2.5 ⫻ 106 PBMC is 75 mU, we could
determine that there was ⬃1% cell death. Regarding isolated PMN
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Ficoll-Hypaque PLUS. After centrifugation at 400 ⫻ g for 35 min at 20°C,
PMN and PBMC appeared in two separate bands.
The lower band containing PMN and erythrocytes was resuspended in
a lysis buffer containing 155 mmol/L NH4Cl, 2.96 mmol/L KHCO3, and
3.72 mmol/L disodium EDTA for 10 min. The cell suspension was then
centrifuged, and PMN were finally resuspended at 50 ⫻ 106 cells/ml in the
culture medium composed of M199, 2 mmol/L glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin.
The upper band contained PBMC. As described earlier (7), PBMC were
washed in 5 mmol/L EDTA-PBS four to six times to remove remaining
platelets. The resulting mononuclear fraction contained less than two platelets per leukocyte. Nonspecific ␣-naphtyl-acetate esterase staining indicated that the mononuclear fraction contained 28.3 ⫾ 6.4% (n ⫽ 4) of
monocytes. PBMC were resuspended at 25 ⫻ 106 PBMC/ml in the culture
medium and were plated (10 ␮L) in sterile 96-well polystyrene tissueculture plates (Nunc, Roskilde, Denmark) containing 100 ␮L of culture
medium. In some experiments, PBMC were preincubated for 15 min with
different pharmacological agents before being plated. These agents included cycloheximide, actinomycin D, NAC, and pyrrolidine dithiocarbamate (PDTC). Then, 10 ␮L of culture medium containing no PMN or
various concentrations of PMN were added. When indicated, PMN were
incubated with polymyxin B for 15 min before being added to PBMC.
PMN and mononuclear cell mixtures always originated from the same
donor.
LPS, obtained from Escherichia coli 0111:B4, was incubated with
PMN/PBMC cocultures at various concentrations (10 ng/ml–10 ␮g/ml). In
selected experiments, PBMC and PMN were cultured separately using an
inner-well system (Nunc Tissue Culture Inserts, 0.2 ␮m anopore membrane; Nunc), in which PBMC were plated in the tissue-culture plate and
PMN were plated in the cell culture inserts. The cell culture inserts containing resting or fMLP-stimulated PMN were removed after various periods of time, from 5 min to 20 h. In some experiments, PBMC were
incubated with H2O2, or X and xanthine-oxidase (XO). In all cases, cells
were cultured at 37°C under 5% CO2/95% air, and the PCA was measured
after 5 or 20 h.
Cell viability, assessed by the measurement of lactate dehydrogenase
(LDH) release in the supernatant of cultured cells (LDH Optimized, Sigma)
and by trypan blue exclusion was ⬎90%. All reagents used for cell isolation and culture were prepared with endotoxin-free water. The levels of
endotoxin contamination in the different reagents incubated with PBMC, as
assessed by a chromogenic Limulus assay (Chromogenix, Mölndal, Sweden), were very low (⬍0.001 ng/ml, final concentration). This level of
endotoxin did not enhance the PCA of PBMC (data not shown).
3823
3824
(5 ⫻ 106 cells/ml), which released 7.16 ⫾ 2.57 mU/ml during the
20-h culture and contain 180 mU/5 ⫻ 106 cells of LDH, the rate
of cell death was higher (4%). When PBMC and PMN were cocultured for 20 h, the measured amount of released LDH (7.64 ⫾ 1.61
mU/ml) was comparable to that expected if the amount of LDH
released by isolated cells was summed (8.14 mU/ml).
In the presence of LPS (10 ng/ml–10 ␮g/ml), the PCA of PMN
was still negligible, whereas that of PBMC increased in a dosedependent manner to level off at 10 times the baseline values at 1
and 10 ␮g/ml of LPS (Fig. 2). The enhancing effect induced by low
concentrations of LPS (10 and 100 ng/ml) was altered when
PBMC were cocultured with PMN; it was further potentiated when
the PMN/PBMC ratio was low (1/5), and it was inhibited when this
ratio was high (2/1). Both the potentiating and inhibitory effects
induced by PMN disappeared at higher concentrations of LPS
(1–10 ␮g/ml).
FIGURE 3. Effect of increasing concentrations of PMN on the TF
mRNA levels of PMN/PBMC cocultures. PMN, PBMC, or PMN/PBMC
cocultures were cultured at 37°C, and after 2, 5, and 20 h, total RNA was
extracted and used for RT-PCR studies with cDNA probes for TF and DR
(n ⫽ 3, 7, and 7, respectively). The relative levels of TF mRNA were
quantified by densitometric scanning and normalized according to the level
of DR mRNA (which was not affected by PMN). PMN did not express
significant levels of TF mRNA or DR mRNA. One representative experiment is shown. Statistical comparisons of the level of TF mRNA of PMN/
PBMC cocultures incubated with increasing concentration of PMN vs that
obtained without PMN are represented by the following: ⴱⴱ, p ⬍ 0.01.
After 5 h of culture, the PCA of isolated PBMC was very low,
even in the presence of LPS (10 ng/ml; data not shown). Similarly,
the potentiating and inhibitory effects of PMN were barely detectable. Therefore, except when indicated, all subsequent experiments
were performed with 20-h cultures.
Finally, in all these experiments, the PCA present both at the
cell surface and in the cell supernatant was identified as TF, as
shown by the fact that ⬎95% was abolished by a cocktail of neutralizing anti-TF mAbs (PCA ⬍ 0.1 AU/106 cells; n ⫽ 1). In
addition, there was no PCA when a factor VII-deficient plasma
was used (PCA ⬍ 0.1 AU/106 cells; n ⫽ 1).
Effect of PMN on TF synthesis and TF mRNA levels by PBMC
The induction mechanism elicited by PMN required de novo protein synthesis. When PBMC were pretreated with actinomycin D
(5 ␮g/ml) or cycloheximide (10 ␮g/ml) for 15 min and then cocultured with PMN for 20 h, the potentiating effect induced by PMN
was completely abolished; at the 1/5 PMN/PBMC ratio, the PCA
of PBMC was 117 and 82% of that of PBMC cultured alone with
actinomycin D and cycloheximide, respectively (n ⫽ 3).
Comparably to the PCA, when cells were treated with increasing
concentrations of PMN, TF Ag levels increased to peak at 1.3-fold
with a PMN/PBMC ratio of 1/5 from 166 ⫾ 38 to 217 ⫾ 31 pg/106
cells (n ⫽ 8). With a PMN/PBMC ratio of 2/1, TF Ag levels were
inhibited compared with those of PBMC cultured alone (105 ⫾ 20
pg/106 cells; p ⬍ 0.01).
TF mRNA transcripts were not detected in PMN cultured alone
for 4 or 20 h but were detectable in PBMC (Fig. 3). At a PMN/
PBMC ratio of 1/5, there was an increase of TF mRNA levels that
was detectable at 2 h, maximal at 5 h, and stable during at least
20 h ( p ⬍ 0.01). At a ratio of 2/1, the TF mRNA levels were
slightly inhibited compared with those of PBMC cultured alone.
Role of PBMC-PMN contact
FIGURE 2. Effect of increasing concentrations of PMN on the PCA of
PMN/PBMC cocultures in the presence of increasing concentrations of
LPS. PMN, PBMC, or PMN/PBMC cocultures were cultured at 37°C and
the PCA expressed on the cell surface was measured after 20 h. Statistical
comparisons of the PCA of PBMC incubated with increasing concentrations of PMN vs that obtained without PMN are represented by the following: ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; n ⫽ 5 or 6.
To ascertain whether direct cell-cell contact and/or secretory products were involved, PBMC and PMN were incubated separately
using an inner-well system for 20 h. At the end of the incubation,
the inserts containing different concentrations of PMN were removed and the PBMC-associated PCA was measured. PMN potently stimulated the PCA of PBMC, and the effect was even more
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FIGURE 1. Effect of increasing concentrations of PMN on the PCA of
PMN/PBMC cocultures. PMN, PBMC, or PMN/PBMC cocultures were
cultured at 37°C, and the PCA expressed on the cell surface was measured
after 20 h. Statistical comparisons of the PCA of PBMC incubated with
increasing concentrations of PMN vs that obtained without PMN are represented by the following: ⴱⴱ, p ⬍ 0.01 for potentiating effect; 00, p ⬍ 0.01
for inhibitory effect; n ⫽ 13.
NEUTROPHIL AND MONONUCLEAR CELL INTERACTIONS
The Journal of Immunology
3825
Table I. Time course of the effect of PMN on the procoagulant activity
of PBMCa
PMN/PBMC Ratio, PCA (%)
marked than when the cells were incubated together (Fig. 4). In
addition, there was no inhibitory effect when the PMN/PBMC cell
ratio was 2/1. Comparable results were found when the level of TF
mRNA transcripts was analyzed; they were increased by 240 ⫾
97% and 197 ⫾ 55% when the PMN/PBMC cell ratio was 1/5 and
2/1, respectively (n ⫽ 4). Thus, contrary to the inhibitory effect,
the potentiating effect of PMN did not require direct cell contact
with PBMC. Therefore, it was induced by a soluble mediator(s)
released by PMN. Thus, when the supernatant of increasing concentrations of PMN plated for 1 h at 37°C was deposited for 20 h
on PBMC, there was a PMN concentration-dependent enhancement of the PBMC PCA that reached 3.7-fold the baseline value
with 5 ⫻ 106 PMN/ml (n ⫽ 1). However, this potentiating effect
was less than that observed when the cells were cocultured using
the inner-well system (21-fold the baseline value with 5 ⫻ 106
PMN/ml; n ⫽ 1), i.e., when the soluble mediator(s) released by
PMN could directly reach PBMC, suggesting that these mediators
were short-lived and partially destroyed during the preparation of
supernatant.
Time course of PBMC stimulation by PMN
We next determined the time required by PMN to maximally stimulate PBMC. PMN were deposited in inserts and cocultured with
PBMC for 5, 15, 30, and 60 min and 20 h, after which the inserts
containing different concentrations of PMN were removed. The
PCA of PBMC was measured at 20 h. Table I shows that a coculture of 30 min was sufficient to make the stimulatory effect of
PMN detectable. At 60 min, the effect was maximum and comparable to that found when the coculture lasted for 20 h.
0.02
0.2
2.0
5 min
15 min
15 min ⫹ fMLP
30 min
60 min
20 h
101 ⫾ 19
111 ⫾ 18
117 ⫾ 26
160 ⫾ 30
156 ⫾ 32
168 ⫾ 31
86 ⫾ 13
129 ⫾ 18
169 ⫾ 25
181 ⫾ 32
226 ⫾ 53
311 ⫾ 73**
107 ⫾ 13
118 ⫾ 15
218 ⫾ 23**
233 ⫾ 46**
331 ⫾ 103**
306 ⫾ 99**
a
PBMC were cultured with increasing concentrations of PMN for various time
periods. PMN were deposited in cell culture inserts for 5, 15, 30, and 60 min and 20 h,
after which the inserts containing PMN were removed. The PCA of PBMC was
measured at 20 h. In one set of experiments, PBMC were cocultured with PMN
stimulated with fMLP (1 ␮mol/L) for 15 min before being removed. Results are
expressed as a percentage of the PCA of PBMC cultured alone for 20 h. Statistical
comparisons of the PCA of PBMC incubated with increasing concentrations of PMN
vs that obtained without PMN are represented by asterisks (ⴱⴱ, p ⬍ 0.01; n ⫽ 4).
nificantly enhanced the PCA of PBMC ( p ⬍ 0.01), which indicates that the PMN stimulation accelerated the release of a soluble
mediator(s) that subsequently acted on PBMC.
Involvement of ROS in PMN stimulation of the PCA of PBMC
The secretory products of PMN include ROS. When PMN were
incubated in polystyrene culture plates in the absence of any exogenous activator, there was a time-dependent and concentrationrelated increase of superoxide generation (Fig. 5), which confirms
that adherence of PMN to polystyrene tissue-culture plates itself
triggers the respiratory burst, even in the absence of any exogenous
agonist (18). The production of superoxide was comparable when
PMN was cultured with and without LPS (8.4 ⫾ 2.2 and 11.9 ⫾
0.8 nmol/ml after 15 min, respectively; p ⬎ 0.05; n ⫽ 3). However, stimulation of PMN with fMLP elicited an acceleration of the
rate of superoxide generation (55.5 ⫾ 2.3 nmol/ml after 15 min;
p ⬍ 0.01 vs nonstimulated PMN; n ⫽ 3).
The role of ROS in the PMN stimulation of the PCA of PBMC
was assessed using the following two antioxidants: NAC, a sulfhydril group donor that acts by increasing levels of the endogenous
antioxidant-reduced glutathione peroxidase system and can directly scavenge free radicals; and PDTC, which scavenges free
radicals and chelates transition metal ions. For example, the total
amount of superoxide anion produced by PMN (5.0 ⫻ 106 PMN/
ml) during 80 min decreased from 30 ⫾ 5 nmol/L to ⬍1 nmol/L
in the presence of NAC (2 mmol/L; n ⫽ 3), as already shown in
a previous study (19). Incubation of PBMC in the presence of
increasing doses of NAC (200 ␮mol/L and 2 mmol/L) or PDTC
Effect of PMN stimulation on the PCA of PBMC
The previous experiments were performed with unstimulated
PMN. In this set of experiments, we determined whether the PMN
stimulation by the chemotactic peptide fMLP (1 ␮mol/L) could
enhance the stimulatory effect of PMN. Resting or fMLP-stimulated PMN were cultured for 15 min with PBMC in a separate
manner using cell-culture inserts, and then were removed. The
PCA of PBMC was measured after 20 h of culture. fMLP did not
enhance the PCA of isolated PBMC. Similarly, the PCA of PBMC
cocultured with nonstimulated PMN for 15 min was not significantly modified (Table I). In contrast, fMLP-stimulated PMN sig-
FIGURE 5. Effect of time on the superoxide anion production (O2⫺) by
increasing concentrations of PMN. Resting PMN were plated in tissueculture plate, and O2⫺ generation was measured for 80 min as the superoxide dismutase-inhibitable reduction of ferricytochrome-c (n ⫽ 3).
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FIGURE 4. Effect of PMN/PBMC contact on the modulatory effect of
PMN on the PCA of PBMC. PBMC were incubated with increasing concentrations of PMN together (No Insert) or separately using an inner-well
system (Insert) for 20 h at 37°C. The PCA expressed on the cell surface
was measured after 20 h. Statistical comparisons of the PCA of PBMC
incubated with increasing concentrations of PMN vs that obtained without
PMN are represented by the following: ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; n ⫽ 8.
Time
3826
NEUTROPHIL AND MONONUCLEAR CELL INTERACTIONS
doses of XO (10 mU/ml) or H2O2 (Fig. 7) resulted in an inhibition
of the PCA.
Discussion
(10 ␮mol/L) resulted in an inhibition of the PMN stimulation of
their PCA without (Fig. 6) or with LPS (data not shown). Finally,
the PMN-mediated stimulation of TF mRNA levels was also abolished by NAC (2 mmol/L) and PDTC (50 ␮mol/L); when the
PMN/PBMC cell ratio was 1/5, they were 110 ⫾ 21% (n ⫽ 7) and
74 ⫾ 32% (n ⫽ 7), respectively, of the level of TF mRNA transcripts measured in PBMC cultured alone.
To confirm that ROS were responsible for the stimulation of
PCA of PBMC, we tested whether incubation of PBMC with
X/XO or H2O2 directly stimulated PBMC. The enzyme system
X/XO was chosen because it produces both O2⫺ and H2O2 (20).
Incubation of PBMC with either XO (1 mU/ml) or X (0.4 mmol/L)
had no effect on their PCA, whereas with both XO and X, the PCA
was increased 5-fold from 4.92 ⫾ 1.28 to 23.0 ⫾ 7.8 AU/106 cells
( p ⬍ 0.05; n ⫽ 5). Likewise, H2O2 alone or with LPS increased
the PCA of PBMC in a dose-dependent manner (Fig. 7). Higher
FIGURE 7. Effect of H2O2 on the PCA of resting or LPS-stimulated
(100 ng/ml) PBMC. PBMC (2.5 ⫻ 106/ml) were cultured at 37°C, and the
PCA expressed on the cell surface was measured after 20 h. Statistical
comparisons of the PCA of PBMC incubated with increasing concentrations of H2O2 vs that obtained without H2O2 are represented by the following: ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; n ⫽ 9.
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FIGURE 6. Effect of NAC or PDTC on the PCA of resting PBMC
(2.5 ⫻ 106 cells/ml) cocultured without PMN (PBMC) or with increasing
concentrations of PMN (0.05 ⫻ 106 cells/ml, PMN/PBMC ratio of 1/50;
0.5 ⫻ 106 cells/ml, PMN/PBMC ratio of 1/5; and 5 ⫻ 106 cells/ml, PMN/
PBMC ratio of 2/1). PBMC or PMN/PBMC cocultures were cultured at
37°C, and the PCA expressed on the cell surface was measured after 20 h.
Statistical comparisons of the PCA of PBMC incubated with increasing
concentrations of PMN vs that obtained without PMN are represented by
the following: ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; n ⫽ 4.
This study demonstrates for the first time that PMN exert concentration-dependent regulatory effects on the expression of the procoagulant protein TF by resting or LPS-stimulated PBMC. First,
cocultures of PBMC with low concentrations of PMN resulted in
a marked increase in TF mRNA, TF protein, and TF activity of
PBMC. Second, when PBMC were cultured with higher concentrations of PMN, the TF expression of PBMC was no longer stimulated but was even inhibited compared with that of PBMC cultured alone.
Different experimental findings indicate that the stimulating effect was mediated through molecules released by PMN; it did not
require direct cell contact between PMN and PBMC (Fig. 4), it
was reproduced with the supernatant of cultured PMN, and it was
enhanced when the PMN release was stimulated by fMLP, a chemotactic peptide (Table I). Among the different potent mediators
released by PMN, we focused our attention on ROS because they
have been shown to induce the expression of TF by endothelial
cells (13). Thus, the stimulating effect induced by PMN was completely prevented when PBMC were preincubated with two structurally unrelated antioxidants, NAC and PDTC (Fig. 6). In addition, the TF expression could be induced by exposing PBMC to
H2O2 (Fig. 7) or to O2 intermediates generated by the X-XO system. ROS have recently gained attention as important second messengers during cell activation. They induce the initiation of gene
transcription and the translocation of transcriptional activating factors into the nucleus (21). The human TF gene contains consensus
sequences for the binding of several transcription factors including
NF-␬B (22). This transcription factor participates in the induction
of TF gene transcription (23, 24). Therefore, because ROS have
been implicated as one of the intracellular second messenger molecules that induce NF-␬B activation (25, 26), one can speculate
that PMN stimulated or potentiated the TF expression of resting or
LPS-stimulated PBMC by the release of ROS, which activated the
TF gene of monocytes through transactivating NF-␬B. However, it
is also possible that PMN ROS acted on other cells or components
present in PBMC, which could lead to TF expression on
monocytes.
Mechanisms involved in the inhibitory effect induced by high
concentrations of PMN appear to be more complex. In contrast to
the previous phenomenon, this inhibitory effect was not reproduced with the cell supernatant, and it required direct cell contact
between PMN and PBMC. However, despite these findings, we
hypothesize that the inhibitory effect also was mediated through
the release of ROS. It previously has been shown that cell exposure
to ROS results in depressed protein synthesis (27). In addition, the
LPS-stimulated expression of TF by PBMC was also inhibited
when PBMC were exposed to increasing doses of H2O2 (Fig. 7). In
this regard, one should note that it is difficult to compare the effect
of ROS released by PMN that acted upon monocytes in a slow and
continuous manner with that induced by purified H2O2 that was
directly added to PBMC. The inhibitory effect was not due to a
PMN-mediated cytotoxicity as shown by the measurement of LDH
in the cell culture supernatant. Furthermore, monocytes cocultured
with PMN for 20 h exhibited normal morphology (data not
shown). In this regard, it is important to note that fresh monocytes
have a high level of hydrogen peroxide-degrading enzymes (28)
and, therefore, that in our experimental conditions their death by
The Journal of Immunology
when both cell populations were separated by a 0.2-␮m filter
(Fig. 4).
Finally, our study describes another situation in which the TF
expression of monocytes is markedly influenced by surrounding
cells through physical contact per se and/or biochemical interactions. Thus, platelets enhance LPS-induced TF activity in monocytes through the production of P-selectin, a cell adhesion molecule (39); platelet factor-4, a platelet ␣-granule (40); and 12hydroxyeicosatetraenoic acid, a metabolic product of the platelet
12-lipoxygenase pathway (41). Likewise, adhesion of monocytes
to activated endothelial cells induces TF generation on monocytes
(42, 43). In another study, granulocytes amplified TF expression
induced by LPS in a platelet-dependent manner (44). In this study,
we show that granulocytes, in the absence of platelets, directly
influence TF expression by PBMC.
In conclusion, this study shows that PMN may play an important
role in the pathogenesis of thrombosis and atherosclerosis by exerting concentration-dependent regulatory effects on the TF production by PBMC. It describes another example where ROS exert
different effects through activation or inhibition of cell functions,
and it indicates a new mechanism by which they may represent a
risk factor for cardiovascular events such as unstable angina and
myocardial infarction, thereby confirming that clinical use of antioxidant in such pathology may be beneficial.
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Cell contact between PMN and PBMC was required for the
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the potentiating effect was less marked when PBMC and low concentrations of PMN were cultured together than when they were
cultured separately (Fig. 4). Thus, we suggest that when PMN and
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Overall, these results suggest that ROS elicit opposite reactions.
This property previously has been shown in other experimental
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NEUTROPHIL AND MONONUCLEAR CELL INTERACTIONS