© 2001 American Heart Association, Inc.
Cellular Biology |
Activation of Human Neutrophil by Cytokine-Activated Endothelial Cells
From the Second Department of Physiology (T.T., F.H., K.S., S.K.), Department of Clinical Hematology (T.Y.), Second Department of Internal Medicine (T.T., Y.N.), and Department of Obstetrics and Gynecology (H.F., S.O.), Osaka City University Medical School, Osaka, Japan.
Correspondence to Tatsuji Takahashi, MD, Second Department of Physiology, Osaka City University Medical School, 1-4-3, Asahi-machi, Abeno-ku, Osaka, 545-8585, Japan. E-mail m8515397{at}med.osaka-cu.ac.jp
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Abstract—Cytokine activation of vascular endothelial cells renders the hyperadhesiveness for neutrophils. During the processes of inflammation and atherosclerosis, the production of reactive oxygen species by neutrophils contributes to endothelial cell (EC) damage and injury. However, the precise mechanisms for neutrophil activation by ECs remain unknown. Thus, we investigated what kinds of pathophysiological factors synthesized by inflammatory cytokine-activated ECs potentiated the activity of neutrophil functions. The magnitude of O2- release from neutrophils, which is one of pivotal neutrophil functions, was measured as an indicator potentiated by activated ECs. Neutrophils release massive amounts of O2- on coculture with activated ECs. Anti–granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody (Ab) or specific platelet-activating factor (PAF)-receptor antagonist suppressed the O2- release from neutrophils on coculture with the activated ECs by 50% to 70%. The supernatants from activated ECs also induced O2- release by neutrophils. This stimulatory effect of activated EC supernatants on O2- release by neutrophils was abolished by anti–GM-CSF Ab or by PAF-receptor antagonist. As we previously reported, we demonstrated the expression of GM-CSF mRNA by Northern blotting and protein synthesis of GM-CSF by ELISA on tumor necrosis factor as well as interleukin-1–activated ECs. Although phosphorylation of mitogen-activated protein kinases was observed in ECs stimulated by tumor necrosis factor and interleukin-1, treatment of ECs with PD98059 (MEK1 inhibitor) and SB203580 (p38 mitogen–activated protein kinase inhibitor) in the presence of the cytokine failed to attenuate the stimulatory effect of activated ECs on neutrophil activation. We found that activated ECs regulated neutrophil function on coculture. We show here for the first time, to our knowledge, that the collaboration between GM-CSF and PAF synthesized by activated ECs markedly potentiated neutrophil activation.
Key Words: endothelial cell • neutrophil • granulocyte-macrophage colony-stimulating factor • platelet-activating factor • intercellular adhesion molecule-1
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Introduction |
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Vascular endothelial cells (ECs) are the gatekeepers of the tissues from the perspective of the bloodstream, and alterations of the endothelium are important determinants in regulating traffic of circulating cells and molecules into various tissues.1 At the sites of inflammation and atherosclerosis, proinflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), and other mediators play crucial roles in cell-cell interactions.2 The cytokine network also plays a role in mediating the host inflammatory response. This response may occur via the increased expression of leukocyte adherence molecules on the surface of ECs as well as through elaboration of chemoattractant substances. Activated ECs may acquire the capacity to perform new functions, and this process is the result of quantitative changes in certain gene products.3 The actions of IL-1 and TNF on ECs that promote leukocyte adhesion and activation are likely to be of importance in the development of inflammatory responses in vitro and in vivo.4 IL-1 or TNF-activated ECs also generate activators of neutrophils, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1, platelet-activating factor (PAF), and IL-8.5 6
TNF and IL-1 could induce strong activation of mitogen-activated protein kinase (MAPK) 3 subtypes of human ECs.7 8 The MAPKs extracellular signal–regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) seem to be central elements of 3 homologous pathways used by mammalian cells to transduce the messages generated by stressing agents as well as growth factors. We have demonstrated that PD98059, which blocks the phosphorylation of ERK1/2 via inhibition of MEK1/2, and SB203580, which blocks the enzyme activity of p38 MAPK, markedly reduced the magnitude of GM-CSF and TNF-induced O2- release by neutrophils.9 Thus, phosphorylation of MAPK may be required for neutrophil activation in response to TNF and GM-CSF.
GM-CSF, a member of the hematopoietic growth factor family, is produced and released by monocytes, macrophages, T cells,10 fibroblasts, vascular smooth muscle cells,11 and ECs in response to IL-112 and TNF.13 In addition to its growth-promoting effects, GM-CSF stimulates a range of functional activities of mature neutrophils,14 15 monocytes, and eosinophils,16 including regulation of leukocyte adhesion, augmentation of surface antigen expression, O2- release, and enhancement or induction of cytokine production.17 GM-CSF produced by ECs plays an important role in the regulation of blood-vessel function.
PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is also a highly potent phospholipid mediator of inflammation and cell-cell interaction.18 19 Newly synthesized PAF on the activated EC surface may act at the beginning of an inflammatory cascade as a mediator that amplifies and propagates the reaction.20 21
Thus, leukocyte activation is a key feature of the progression associated with atherosclerosis as well as inflammation. However, little is known about the role of interaction between ECs and neutrophils in the development of inflammation and atherosclerosis. The magnitude of O2- release from neutrophils, as an indicator of the respiratory burst, was considered to be a marker potentiated by ECs. In the present study, we investigated what kinds of mediators synthesized by ECs caused the neutrophil activation.
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Materials and Methods |
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Reagents and Cells
PAF (C16: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), cytochrome c type III, superoxide dismutase, 3,7-dimethyl-1-propargylxanthine (DMPX), and adenosine deaminase (ADA) were obtained from Sigma Chemical. Highly purified recombinant human GM-CSF, TNF-
, and IL-1ß produced by
Escherichia coli were provided
by Schering-Plough, Dainippon Pharmaceutical, and Otsuka
Pharmaceutical, respectively. Polyclonal sheep anti-human GM-CSF
antibody (Ab) was provided by Schering-Plough. PD98059 and rabbit
polyclonal antibodies against
Thr202/Tyr204-phosphorylated
ERK1/2,
Thr180/Tyr182-phosphorylated
p38, and
Thr183/Tyr185-phosphorylated
JNK1/2 were obtained from New England Biolabs. SB203580 was provided by
SmithKline Beecham Pharmaceuticals. Specific PAF receptor antagonists,
YM264 and WEB2170, were gifts from Yamanouchi Research Institute
(Osaka, Japan) and Boeringer Ingelheim (Ingelheim, German),
respectively. Human ECs from umbilical cord vein, prepared and
characterized as previously
described,22 were grown on
2.5% gelatin-precoated 60-mm tissue culture petri dishes (Nunc). The
immunofluorescent staining for factor VIII was confirmed. Neutrophils
were prepared as described.9
The final cell concentration was 6x106
cells/mL.
Activated EC-Neutrophil Interaction and
O2- Release
Assay
Confluent EC monolayers at passages 2 through 5 were
removed from the dishes, and 1.5x105 ECs
were seeded onto gelatin-precoated 24-well plates. TNF and IL-1 were
added to the wells at the indicated concentrations, and the plates were
incubated for 4 hours at 37°C. The wells were gently washed 3 times
with warmed HBSS. When TNF and IL-1–pretreated EC monolayers were
fixed with 1% paraformaldehyde, the fixed ECs were thoroughly washed 3
times. Reaction buffers (0.2 mL) were added to each well. Then
neutrophils (6x105 cells)
were placed in contact with EC monolayers (0.4 mL/well). When
supernatants from activated ECs or coculture were required, the buffers
(0.2 mL/well) were added alone. If the supernatants, cytokine, or
mediator-induced
O2- release by
neutrophils were measured, neutrophils
(3x105 cells) were put on
FCS-precoated 48-well plates. The plates were incubated for 3 hours at
37°C. The wells were aspirated, and the supernatants were centrifuged
to remove cells. As previously
described,9 23 the
O2- release was
determined as superoxide dismutase–inhibitable reduction of
ferricytochrome c. Results from
duplicate wells were averaged.
Intercellular Adhesion Molecule-1
Expression Study
Confluent ECs treated with or without IL-1 and TNF at
the indicated concentrations were harvested at the indicated time and
incubated for 30 minutes at 4 °C with either monoclonal Ab (84H10)
directed against intercellular adhesion molecule-1 (ICAM-1) conjugated
with FITC or IgG1 (Sigma Chemical) as control Ab. Membrane antigen
expression of activated ECs was analyzed by a flow cytometer
(FACScalibur, Becton Dickinson).
Western Blotting
As previously
described,9 human umbilical
vein ECs (HUVECs) were stimulated by cytokines for the indicated time.
The blots were incubated with appropriate Ab. The membrane was
incubated with anti-rabbit lgG Ab conjugated with horseradish
peroxidase, and the Ab complexes were visualized using the ECL
detection system as directed by the manufacturer.
Statistical Analysis
Results are presented as mean±SEM. Differences
between groups were analyzed with unpaired Student’s
t test.
P<0.05 was considered
significant.
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Results |
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Cytokine-Activated EC-Neutrophil Interaction
Our results showed that activated ECs potentiate massive amounts of O2- release from neutrophils (Figure 1A
). This indicates that activated ECs augmented the
activity of neutrophil function. The activity of ECs treated with the
combination of IL-1 (8.19±1.17
nmol/6x105cells per 3 hours) and TNF
(10.82±1.49 nmol/6x105cells per 3 hours)
was subadditive (13.86±3.64
nmol/6x105cells per 3 hours). Our results
demonstrated that the amount of
O2- release from
neutrophils on the coculture was dependent on the concentration of IL-1
or TNF used to activate ECs
(Figure 1B). IL-1–treated ECs evoked neutrophil activation
at low concentration and rapidly reached the plateau, whereas
TNF-treated ECs slowly caused neutrophil activation. The
O2- release
gradually increased with time (data not shown). In our assay, the
amount of O2-
release in neutrophils stimulated by TNF- (100 U/mL=39.2 ng/mL) or
GM-CSF (5 ng/mL), which is the optimal concentration, was 5.86±0.35 or
4.19±0.26 nmol/3x105cells per 3 hours,
respectively.9 ECs did not
release O2- in
response to these cytokines.
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5 separate experiments, each done in duplicate. *P<0.05 and **P<0.01 compared with CTL.
Supernatants From Activated ECs or Coculture
Induced O2- Release
by Neutrophils
The supernatants from activated ECs alone could induced
O2- release by
neutrophils (IL-1, 2.47±0.35; TNF, 3.21±0.43; and IL-1+TNF,
4.77±0.92 nmol/3x105cells per 3 hours) as
potent as the supernatants from coculture (IL-1, 2.25±0.31; TNF,
3.42±0.54; and IL-1+TNF, 4.92±0.98
nmol/3x105cells per 3 hours)
(Figure 2). The supernatants on coculture with neutrophils
did not synergistically augment the amount of
O2- release from
neutrophils in comparison with
monocytes.22
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Phosphorylation of MAPK 3 Subtypes in
IL-1–Activated or TNF-Activated ECs
The results presented in
Figure 3 show that all 3 MAPKs (ERK1/2, p38, and JNK1/2) in
ECs were phosphorylated in response to IL-1 (1.25 ng/mL) or TNF (39.2
ng/mL) in agreement with previous
studies.7 8 Both
IL-1 and TNF activated ERK1/2, p38, and JNK1/2 in
ECs.
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Neutrophils Adherent to Cytokine-Activated
ECs
After 5 minutes of coculture, nonadherent neutrophils
were removed. Neutrophils bound to ECs were washed 3 times with warmed
HBSS and fixed with 1% paraformaldehyde. The number of neutrophils
adherent to ECs was counted using a phase-contrast microscope (Nikon
Diaphot). The number of neutrophils was markedly increased in adherence
to activated ECs (IL-1, 34.3±5.8; TNF, 52.2±8.6; and IL-1+TNF,
82.6±10.4 cells/field), as shown in
Figure 4A. Furthermore, we analyzed ICAM-1 expression on the
surface of IL-1–activated or TNF-activated ECs by FACS, and ICAM-1
expression on IL-1–activated or TNF-activated ECs was upregulated in a
dose- and time-dependent manner in agreement with previous studies
(Figures 4B and 4C).24
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We next investigated the involvement of ICAM-1–mediated
signaling events during neutrophil adherence in the increase of
O2- release from
neutrophils. ECs were fixed to prevent the formation of de
novo–generated EC mediators in accordance with previous
studies.16 25 26
In agreement with previous studies, the ability of viable neutrophils
to undergo adhesion in response to cytokines was not impaired in the
presence of fixed ECs (data not shown). Cytokine-treated fixed ECs had
higher levels of adhesion molecules, including ICAM-1, on their
surfaces than control-fixed ECs
(Figures 4B
and 4C). The magnitude of
O2- release from
neutrophils on incubation with the cytokine-treated fixed ECs in the
presence of activated ECs supernatants was significantly greater than
that on incubation with nontreated fixed ECs
(Figure 5A). Cytokine-treated fixed ECs also augmented the
recombinant human (rh) GM-CSF (50 or 500 pg/mL)–induced
O2- release by
neutrophils but not other doses of rh GM-CSF (5 pg/mL and 5 ng/mL)
(Figure 5B). However, this enhancement of adhesion to
activated ECs in
O2- release by
neutrophils was small. On the other hand, eosinophil adhesion to VCAM-1
markedly enhanced
O2-
release.16 26 The
amount of O2-
release from neutrophils in contact with TNF-activated fixed ECs
gradually increased in a manner dependent on the dose and time used to
activate ECs (data not shown).
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P<0.05 compared with the absence of rh GM-CSF. ¶P<0.05 compared with CTL-fixed ECs.
The results shown in
Figures 5A and 5B indicate that the amount of
O2- release from
neutrophils induced by activated-EC supernatants was identical with
that induced by nonglycosylated GM-CSF (50 pg/mL) from
E. coli. Because the
stimulatory effect of rh GM-CSF from E.
coli was greater than that of GM-CSF from mammalian cells on
a molar basis,14
nonglycosylated GM-CSF (50 pg/mL) from E.
coli is equivalent to glycosylated GM-CSF (500 pg/mL) from
HUVECs. Taken together, these results suggest that cytokine-activated
ECs might release an equivalent of 
500 pg/mL GM-CSF present in
supernatants.
Effects of Various Inhibitors on
EC-Neutrophil Interaction and Supernatant-Induced
O2- Release
Assays
Neutralizing Ab against GM-CSF (55 µg/mL) and a
specific PAF receptor antagonist (YM264
2.5x10-5
mol/L) inhibited the amount of
O2- release from
neutrophils on coculture with activated ECs by 60% to 70% and, to a
lesser degree, by 50% to 60%, respectively
(Figure 6A). The inhibitory effects of PAF receptor
antagonists (YM264 and WEB2170) on the
O2- release were
dependent on the concentration, and YM264 was more potent than WEB2170,
in agreement with a previous study (data not
shown).27 Anti–GM-CSF Ab
and YM264 almost completely abolished the
O2- release from
neutrophils induced by activated EC supernatants
(Figure 6B). It is suggested that supernatants from
cytokine-activated ECs contain soluble factors that are predominantly
GM-CSF and PAF. Neither YM264 nor WEB2170 had any effect on control or
TNF-primed
N-formyl-methionyl-leucyl-phenylalanine–induced
O2- release by
neutrophils.
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Because IL-1 and TNF activated MAPK, as shown in
Figure 3, we investigated the involvement of MAPK cascade in
ECs in neutrophil activation. We have demonstrated that PD98059 and
SB203580 reduced the magnitude of GM-CSF–induced and TNF-induced
O2- release by
neutrophils.9 When PD98059
and SB203580 were added to EC monolayers 15 minutes before the
treatment with IL-1 or TNF, treated for 4 hours, and then washed, the
amounts of O2-
release on the coculture between PD98059- and SB203580-treated
activated ECs and neutrophils for 3 hours were not altered in
comparison with the coculture between activated ECs and neutrophils
(data not shown). Although both PD98059 and SB203580 suppressed the
O2- release by
neutrophils on the coculture, in agreement with our previous
study9
(Figure 6A), the pretreatment of activated ECs with both
PD98059 and SB203580 had no significant effect on the activity of
neutrophil function. The results indicate that MAPK cascade might have
an association with neutrophil activation but not with EC activation in
response to inflammatory cytokines.
It is well established that ECs exposed to hypoxic
conditions have an altered
phenotype,28 as demonstrated
by increase in adenosine production. Adenosine inhibits priming of
neutrophils and the respiratory burst of cytokine-triggered adherent
neutrophils via A2
receptor.29 To test whether
adenosine derived from ECs attenuates neutrophil activation, we
examined the effect of 2 adenosine inhibitors, DMPX, which is an
A2-specific receptor antagonist, and ADA, which
can deaminate adenosine to inosine, on the
O2- release from
neutrophils on coculture. As shown in
Figures 6A and 6B, the inhibitory effects of adenosine were
partially but not significantly blocked by both DMPX
(2.5x10-5
mol/L) and ADA (1.25 ng/mL). These data indicate that adenosine did not
mediate the activity of neutrophil function by cytokine-treated ECs
compared with ischemia/reperfusion-treated ECs. Blocking monoclonal Ab
against ICAM-1 (84H10, Immunotech) did not significantly inhibit the
O2- release from
neutrophils on the coculture (data not shown).
GM-CSF Production of Activated ECs
HUVECs spontaneously released low levels of GM-CSF
(16.9±2.7 pg/mL) under control conditions. The concentrations of
GM-CSF produced by IL-1ß (1.25 ng/mL) and TNF- (39.2
ng/mL)–activated ECs were 317.6±22.8 and 421.5±39.4 pg/mL,
respectively. When ECs were treated with the combination of IL-1 and
TNF, the additive response in the production of GM-CSF from activated
ECs was observed (758.3±81.4 pg/mL). The levels of GM-CSF production
by activated ECs in the presence of SB203580
(1x10-6 mol/L)
were higher than those in the absence of SB203580; however, there were
no significant differences of GM-CSF production by IL-1–activated and
TNF-activated ECs in the presence or absence of SB203580 or PD98059.
Similar to ICAM-1 expression on the surface of ECs, the levels of
GM-CSF production by activated ECs were dependent on cytokine
concentration used to activate ECs (data not shown). The coculture
between IL-1ß or TNF-–activated ECs and neutrophils had no
significant increase in GM-CSF production (data not
shown).
Effect of PAF on GM-CSF–Induced
O2- Release From
Neutrophils
When varying concentrations of exogenous PAF were added
in the presence of rh GM-CSF (50 pg/mL), PAF affected nonglycosylated
rh GM-CSF (50 pg/mL)–induced
O2- release by
neutrophils, which is equivalent to the concentration of glycosylated
GM-CSF from activated ECs. The maximum enhancement of rh GM-CSF (50
pg/mL)–induced O2-
release was elicited by 20 to 50 nmol/L PAF, which was the
physiological concentration of PAF synthesized by activated ECs
(Figure 8A).30
Interestingly, the enhancement of GM-CSF–induced
O2- release by
exogenous PAF (20 nmol/L) was not obtained at higher level of rh GM-CSF
(500 to 5000 pg/mL) compared with the physiological level of GM-CSF
produced by activated ECs
(Figure 8B).31 32
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IL-1 and TNF have multiple actions on ECs that seem to promote both inflammatory and coagulation responses. Although many TNF activities are indistinguishable from those of IL-1, TNF and IL-1 activities are often additive and always seem to be independent (Figures 1A
, 2, 3A, and 7).1 The
cytokines may promote injury and angiogenesis through indirect actions
dependent on leukocytes. Through its effect on major histocompatibility
complex (MHC) antigen expression, TNF may promote lymphocyte-dependent
inflammation (an action not shared by IL-1). Inflammatory mediators are
not always secreted; in some cases, they may be expressed on the cell
surface of the stimulated cells. However, whether secreted or
surface-expressed, the mediators share a common mode of action: they
bind to specific protein receptors on the surface of their target
cell.
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TNF and IL-1 could evoke strong activation of MAPK 3
subtypes in HUVECs. ERK is involved in the regulation of
transcriptional and translational activities and is essential for
control of cell growth and differentiation. p38 MAPK and JNK/SAPK are
recognized as stress-sensitive kinases. Our studies indicated a
parallel pathway in ECs leading to activation of the p38 MAPK and
JNK1/2 in a fashion similar to ERK1/2 activation in response to TNF and
IL-1
(Figure 3).7 8
Two MAPK inhibitors, both PD98059 and SB203580, had no significant
influence on GM-CSF production of the activated ECs
(Figure 7), whereas PD98059 and SB203580 markedly inhibited
the O2- release
from neutrophils.9 Although
both EC and neutrophil activation have common cascade via ERK and p38
MAPK in response to inflammatory cytokines, the degree of contribution
to EC activation via MAPK signaling might be distinct from that to
neutrophil activation, or JNK/SAPK might have an important pathway in
EC activation in response to TNF and IL-1.
ECs produce PAF in response to TNF and IL-1,18 19 20 whereas thrombin, H2O2, histamine, leukotrienes, and phorbol 12-myristate 13-acetate also trigger PAF production.33 Endothelial PAF remains mainly cell-associated and can be recognized by neutrophils that come into contact with the ECs.21 EC-associated PAF is known to participate in regulating neutrophil attachment to activated ECs.34 PAF cooperates with P-selectin and E-selectin in mediating increased neutrophil-EC adherence by triggering an increase in affinity of CD11b/18 (Mac-1).35 36 ECs express ICAM-1 (CD54), a counter-ligand for CD11/18 integrin37 that promotes adhesion and transendothelial migration of neutrophils.38 IL-8, also a potent priming agent for O2- release and phospholipase A2 activation, is known to be produced by TNF or IL-1–treated ECs34 ; however, Hill et al27 have shown that neutralizing Ab against IL-8 was unable to prevent priming of neutrophil function.
In our previous study,22 we demonstrated that GM-CSF mRNA and protein were synergistically expressed on coculture between activated ECs and monocytes, and TNF or IL-1–activated ECs synthesized GM-CSF with maximal production at 4 hours. GM-CSF stimulates a range of functional activities of neutrophils, including regulation of adhesion, augmentation of surface antigen expression, O2- release, and enhancement or induction of cytokine production.39 Thus, it is possible that GM-CSF may contribute to the pathophysiological events involved in atherosclerosis and inflammation. The ability of neutrophils to release large amounts of O2- in response to GM-CSF is presumably controlled by the known receptors for the cytokine.40 GM-CSF also stimulated not only O2- release but also PAF synthesis from neutrophils with time and dose relationships.19 41
Neutrophils are the first inflammatory cells to appear at the site of vessel damage, exacerbating and propagating the inflammatory response. It has also been reported that neutrophils generate reactive oxygen species, which may cause lipid oxidation and lead to serious derangements of cell metabolism, including DNA destruction and modification of proteins,11 thereby contributing to the pathogenesis of atherosclerosis. Neutrophils also possess a variety of granule proteases suggested to contribute to inflammatory tissue injury.42 The increased adhesion and migration of neutrophils through the endothelium are important events in tissue injury.
The response of O2- release by neutrophils is suppressed by EC-derived adenosine, which possesses anti-inflammatory potential and is most likely mediated through the A2 receptor.43 Therefore, ECs regulated neutrophil activation in the process of inflammation. However, it is unlikely that adenosine from cytokine-activated ECs is associated with neutrophil activation potentiated by activated ECs.
We demonstrated that collaboration between GM-CSF and PAF at physiological concentration is essential to enhance neutrophil activation. The results from both a little enhancement of the O2- release by neutrophil adherence to activated ECs and no significant reduction of the O2- on coculture in the presence of anti-ICAM-1 Ab were obtained. Taken together, soluble factors produced by activated ECs, rather than Mac-1–ICAM-1–mediated signaling events during neutrophil adherence, induced neutrophil activation. The selective synthesis of PAF and GM-CSF by activated ECs may play a role in regulating and amplifying the priming and activation of neutrophils on the EC-neutrophil interaction. ICAM-1–ß2 integrin–dependent signaling pathways in neutrophil activation had a minor effect on neutrophil O2- release on the coculture in our assay. Thus, we speculated that adhesion of neutrophils to activated ECs is unlikely to be the main mode of cytokine-activated EC potentiation of neutrophil.
Moreover, we found that the most enhanced effects of both
exogenous PAF (20 nmol/L) and adhesion to cytokine-treated fixed ECs on
O2- release by
neutrophils were evoked by 50 pg/mL of nonglycosylated GM-CSF
(Figures 5B and 8B). This concentration of GM-CSF was
identical to that produced by activated ECs. On the basis of these
findings, we speculate that, first, neutrophil recruitment via
mediators and cytokines produced by activated ECs, such as PAF and
IL-8, is an initial step; second, activated EC-associated PAF primes
neutrophils adherent to ECs via the Mac-1–ICAM-1 pathway; and, third,
potentially more GM-CSF synthesized by activated ECs augmented
neutrophil activation during the transendothelial
migration.
In conclusion, neutrophils primed by activated EC-associated PAF may be strongly potentiated by activated EC-synthesized GM-CSF during the transmigration in vivo. GM-CSF and PAF might be major contributors to neutrophil activation, whereas the Mac-1–ICAM-1 pathway might be a minor contributor. We have demonstrated for the first time, to our knowledge, that in collaboration with activated EC-derived PAF, GM-CSF synthesized by activated EC induced large amounts of O2- release from neutrophils and, furthermore, that cooperation of both GM-CSF and PAF at physiological concentrations was required for augmented neutrophil activation.
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Acknowledgments |
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This study was supported by Grants-in-Aid from the Ministry of Education, Science and Culture, Japan. The authors thank Dr Daisuke Tachibana, Department of Obstetrics and Gynecology, Osaka City University Medical School, for technical assistance.
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Footnotes |
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Original received July 17, 2000; resubmission received December 19, 2000; accepted January 2, 2001.
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References |
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