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Physiology Program, Department of Environmental Health, Harvard School of Public Health, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-activated pulmonary microvascular
ECs induced an increase in the apparent stiffness of ECs within 2 min,
measured with magnetic twisting cytometry. An anti-ICAM-1 Ab
blocked the EC stiffening response without inhibiting neutrophil
adherence. Moreover, cross-linking ICAM-1 mimicked the stiffening
response induced by neutrophils. The neutrophil-induced increase in the
apparent stiffness of ECs was inhibited with 1% DMSO (a hydroxyl
radical scavenger), allopurinol (a xanthine oxidase inhibitor), or
deferoxamine (an iron chelator), suggesting that ROS may be involved in
mediating the EC stiffening response. The cellular sources of ROS were
determined by measuring the oxidation of dichlorofluorescein.
Neutrophil adherence to TNF--activated ECs induced ROS production
only in ECs, and not in neutrophils. This ROS production in ECs was
completely prevented by the anti-ICAM-1 Ab and partially inhibited
by allopurinol. These results suggest that ICAM-1-mediated signaling
events during neutrophil adherence may activate xanthine oxidase, which
in turn mediates the ROS production in ECs that leads to stiffening.
ROS generated in ECs on neutrophil adherence appear to mediate
cytoskeletal remodeling, which may modulate subsequent inflammatory
responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies have provided considerable evidence that not only does neutrophil-EC adherence provide physical interactions between these two cell types but it also results in intracellular signaling in both neutrophils and ECs that may modulate neutrophil emigration. For instance, chemoattractant-induced neutrophil adherence and emigration induce intracellular Ca2+ increases (3), F-actin stress fiber formation (4), myosin light chain kinase activation (4, 5, 6), and isometric tension generation (6) in ECs. Inhibition of these changes in ECs reduces neutrophil emigration across the EC monolayer in vitro (3, 4, 5). Moreover, neutrophil adherence to IL-1-activated ECs leads to intracellular Ca2+ increases and F-actin stress fiber formation in ECs (7). Adhesion also induces signaling in neutrophils through integrins, because neutrophil adherence to surface-bound ß2 or ß3 integrin ligands, including anti-ß2 or anti-ß3 integrin Abs, ICAM-1, or extracellular matrix proteins, results in reorganization of the actin-based cytoskeleton and ROS production (8, 9, 10).
This study evaluated the effect of neutrophil adherence to human pulmonary microvascular ECs on the functions of these ECs, particularly their biomechanical properties. These properties were measured using magnetic twisting cytometry (11, 12). This technique measures the angular rotation (strain) of ferromagnetic beads bound to cells upon application of a magnetic torque (stress), and the apparent stiffness of the cells is defined as the ratio of stress to angular strain. The roles of ICAM-1, reactive oxygen species (ROS), and nitric oxide, in neutrophil-mediated EC stiffening were examined. Finally, the contribution of ICAM-1-mediated adhesion to the production of ROS within ECs was evaluated. These studies showed that neutrophil adhesion to ECs resulted in production of ROS in ECs that was ICAM-1-dependent and that these ROS were required for neutrophil-induced increase in the apparent stiffness of ECs. This pathway may contribute to the regulation of neutrophil emigration and EC permeability.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was obtained from R&D Systems (Minneapolis, MN); murine anti-human
ICAM-1 Ab (clone RR1/1) used as a blocking Ab was obtained from
Biosource International (Camarillo, CA); murine anti-human ICAM-1
Ab (clone 6.5B5) used for cross-linking and ELISA studies and rabbit
anti-mouse IgG Ab were obtained from Dako (Carpinteria, CA); murine
anti-human ICAM-1 Ab (clone LB2) was obtained from Becton Dickinson
(Franklin Lakes, NJ); murine anti-human ß1
integrin Ab (clone P5D2) and murine anti-human P-selectin Ab (clone
P8G6) were obtained from Chemicon (Temecula, CA); murine anti-human
E-selectin Ab (clone 68-5H11), murine anti-human HLA-A,B,C Ab
(clone G46-2.6), and murine IgG (clone MOPC-21) were obtained from
PharMingen (San Diego, CA), murine anti-human CD18 Ab was obtained
from Ancell (Bayport, MN), fluorescein-conjugated goat anti-murine
IgG was obtained from Organon Teknika (Durham, NC). Isolation of human neutrophils
Blood was drawn from healthy humans by venipuncture after informed consent was obtained. Human neutrophils were isolated with Histopaque density gradients (Sigma) according to manufacturer’s protocols. The purity of isolated neutrophils was >95%.
Cultivation of human pulmonary microvascular ECs
Human pulmonary microvascular ECs were obtained from Clonetics
(Walkersville, MD) and plated onto fibronectin-coated culture dishes
according to manufacturer’s protocols. ECs were used between passage 6
and 10. All experiments were performed using cells 3–6 days after they
reach confluence. These cells constitutively express ICAM-1, and they
can be induced to express E-selectin and up-regulate ICAM-1 expression
on TNF- stimulation (13).
Experimental protocol
ECs treated with buffer or TNF- for 24 h were studied.
Cells were cultured in 96-well plates that contained 
40,000
cells/well when the cells reached confluence. For each of the
experiments, an average response from all the cells in a cell well was
recorded, and the n values in this study refer to the number
of individual experiments. After the baseline biomechanical properties
or dichlorofluorescein (DCF) fluorescence of ECs were measured,
neutrophils (neutrophil:EC, 1:1) or buffer were added to ECs, and these
parameters as well as neutrophil shape were measured after 2, 6, 10,
and 15 min of neutrophil adhesion. In many studies, neutrophils or ECs
were pretreated with agents before the adhesion of neutrophils as
described in Results to examine the mechanisms important in
the observations.
Biomechanical properties of ECs
The biomechanical properties of ECs were measured by magnetic
twisting cytometry. This technique measures the angular rotation of
ferromagnetic beads bound to cells through specific ligands on
application of a magnetic torque (stress). The degree of angular
rotation is inversely proportional to the stiffness of the cells to
which the ferromagnetic beads are bound. Ferromagnetic beads coated
with goat anti-mouse IgG (Fc) were obtained from Spherotech
(Libertyville, IL). These beads were incubated with a murine Ab against
human ß1 integrin at a concentration of 1
µg/106 beads for 30 min at 4°C, followed by
three washes in PBS. ECs treated with 20 ng/ml TNF- or buffer for
24 h at 37°C were washed twice with DMEM containing 5% FBS and
incubated with anti-ß1 integrin Ab-coated
beads at 37°C for 30 min. The unbound beads were gently washed off,
and the well was placed in the magnetic twisting cytometer. As
previously described (11, 12), the bound beads were
exposed to a brief (10-µs) but strong (1000-gauss) magnetic field,
which magnetizes the beads in the horizontal direction. After 20
s, the beads were twisted by a much weaker (30-gauss) but continuous
(1-min) vertical magnetic field. This twisting field was not strong
enough to remagnetize the beads, but it caused the beads to rotate. The
magnitude of magnetic vector in the horizontal direction (remnant
magnetic field) was measured by an in-line magnetometer. From this
value, the average bead rotation (angular strain) was calculated
(11, 12). The rotational stress was calculated by rotating
the beads in a viscous standard. For these beads, a twisting field of
10 gauss corresponded to an applied torque at the start of the twist
(initial stress) of 7 dynes/cm2. The specific
torque (stress) on the beads at the end of the 1-min twist
(stress1 min) was calculated with the use of the
initial stress times the ratio of remnant field at the end of the 1-min
twist and the remnant field at time 0. The apparent stiffness was
measured at 1 min of twist and was defined as the ratio of
stress1 min to the angular strain at this time
point.
Measurement of the expression of EC adhesion molecules
The expression of EC adhesion molecules was quantified by ELISA
as previously described (26). Briefly, ECs treated with
buffer or 20 ng/ml TNF- were fixed with 0.1% paraformaldehyde for
20 min at room temperature and washed. The cells were incubated with
the blocking solution containing 1% BSA and 1% goat serum for 30 min,
after which the cells were incubated with 10 µg/ml primary Ab against
ICAM-1, P-selectin, E-selectin, or isotype control Ab diluted in
blocking solution for 1 h. The cells were washed and incubated in
10 µg/ml fluorescein-conjugated secondary Ab for 1 h. After
three washes, the fluorescence (excitation wavelength, 490 nm; emission
wavelength, 530 nm) was quantified using a fluorescent plate
reader.
ICAM-1 cross-linking
ICAM-1 was cross-linked as previously described
(14). Briefly, 24-h TNF-- or buffer-treated ECs were
washed once with DMEM containing 5% FBS and incubated with 15 µg/ml
murine anti-human ICAM-1 Ab (clone 6.5B5), murine anti-human
HLA-A,B,C Ab (clone G46-2.6), or murine IgG for 30 min. The cells were
washed twice, and rabbit anti-murine IgG Ab was added at 1:100
dilution from the manufacturer’s stock. The apparent stiffness of ECs
was measured 2–15 min later.
Neutrophil adhesion assay
Isolated neutrophils were labeled with sodium
[51Cr]chromate as previously described
(15). Confluent ECs plated onto 96-well plates were
treated with 20 ng/ml TNF- for 24 h. After two washes, ECs were
incubated with 50 µg/ml murine anti-human ICAM-1 Ab (clone RR1/1)
or murine IgG for 30 min and washed twice with HBSS containing 1.2 mM
Ca2+, 0.4 mM Mg2+, and 5.5
mM glucose. To examine the role of CD18 in mediating neutrophil
adhesion, neutrophils were incubated with 10 µg/ml anti-CD18 Ab
(clone IB4) for 20 min before being added to ECs.
51Cr-Labeled neutrophils (neutrophil:EC, 1:1)
were then added to the EC wells or to plastic wells containing 100 µl
HBSS along with 1.2 mM Ca2+, 0.4 mM
Mg2+, and 5.5 mM glucose and allowed to adhere
for 15 min. Nonadherent neutrophils were then washed off, and the
fraction of neutrophils that remained adherent was calculated.
Changes in neutrophil shape on adherence to ECs
ECs were grown to confluence on glass coverslips and treated
with 20 ng/ml TNF- or buffer for 24 h. Neutrophils were added
to ECs and incubated for 6, 15, and 30 min. The cells were fixed and
examined under a microscope with a drawing tube. To examine the
projected area and shape of neutrophils, the neutrophils were outlined
on a digitizing pad interfaced with a computer equipped with SigmaScan
software. The projected area, as well as the length of the major axis
and the axis perpendicular to the major axis at its midpoint (the minor
axis), was measured for each cell. The ratio of the major to minor axis
was calculated to evaluate the changes in neutrophil shape that
occurred over time.
Measurement of oxidant production in neutrophils and ECs
To evaluate ROS production in neutrophils, isolated neutrophils
were incubated with 20 µM DCFDA for 30 min at room temperature and
washed twice with Ca2+- and
Mg2+-free HBSS. ECs treated with 20 ng/ml TNF-
or buffer for 24 h were washed twice with HBSS containing 1.2 mM
Ca2+, 0.4 mM Mg2+, and 5.5
mM glucose before labeled neutrophils or buffer were added. DCF
fluorescence was excited at 490 nm, and the emission was collected at
525 nm with a fluorescent plate reader. Neutrophil DCF fluorescence was
measured 0–15 min after adherence to ECs. Then 1 µM fMLP or buffer
was added to the well, and neutrophil DCF fluorescence was measured
2–15 min later.
To evaluate ROS production in neutrophils adherent to plastic, neutrophils were added to plastic wells containing 100 µl HBSS along with 1.2 mM Ca2+, 0.4 mM Mg2+, and 5.5 mM glucose. After the baseline DCF fluorescence was measured, 1 µM fMLP or buffer was added, and the fluorescence was measured 2–15 min later.
To evaluate oxidant production in ECs, ECs treated with 20 ng/ml
TNF- or buffer for 24 h were incubated with 20 µM DCFDA for
45 min at 37°C and washed twice with HBSS containing 1.2 mM
Ca2+, 0.4 mM Mg2+, and 5.5
mM glucose. After the baseline DCF fluorescence was measured,
neutrophils or buffer were added, and the DCF fluorescence was measured
2–20 min later.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-treated ECs
on neutrophil adherence and the roles of ICAM-1 and ROS
The biomechanical properties of ECs were evaluated by magnetic
twisting cytometry with the use of beads coated with
anti-ß1 integrin Ab as described previously
(11, 12). ß1 integrin was selected
because it is a transmembrane protein that is linked to the
cytoskeleton. Pulmonary microvascular ECs expressed
ß1 integrin as determined by ELISA, and TNF-
treatment did not alter its expression (data not shown). Preliminary
studies indicated that the biomechanical properties measured with beads
coated with either of two anti-ß1 integrin
Abs (clones JB1A and P5D2) were similar (apparent stiffness, 18.6 and
19.1 dynes/cm2, respectively). All subsequent
experiments in this study were performed with clone P5D2. When beads
coated with a control mouse IgG were used, the stiffness of ECs was
5.5 ± 1.3 dynes/cm2 (n =
4). These studies demonstrated that binding beads to ECs through
cytoskeleton-linked ß1 integrin decreased their
ability to rotate compared with beads bound nonspecifically. Moreover,
treatment of these ECs with 1 µg/ml cytochalasin D for 30 min reduced
the apparent stiffness of ECs from 19.8 ± 0.7
dynes/cm2 to 12.0 ± 0.9
dynes/cm2 (n = 4), demonstrating
that the integrity of the actin cytoskeleton contributed to the
biomechanical properties of ECs measured with beads coated with
anti-ß1 integrin Ab.
Neutrophil adherence led to changes in the biomechanical properties of
cytokine-activated ECs. Neutrophil adherence for 2 min induced an
increase in the apparent stiffness of 24-h TNF--treated ECs when
measured using ferromagnetic beads bound to ß1
integrin on ECs (Fig. 1
). This increase
in the apparent stiffness of ECs required TNF- treatment, because
neutrophils adherent to untreated ECs did not increase EC stiffness
(Fig. 1). Pretreatment of neutrophils with a blocking anti-CD18 Ab,
which reduced the percentage of neutrophils adherent to 24-h
TNF--treated ECs from 24.2 ± 2.2% to 2.0 ± 1.5%,
significantly inhibited this stiffening response. Adherence of
neutrophils treated with a control mouse IgG to ECs for 2, 6, and 10
min increased the EC stiffness from 22.0 ± 1.6
dynes/cm2 to 26.6 ± 1.3, 34.2 ± 3.0
and 32.3 ± 1.5 dynes/cm2, whereas on
adherence of neutrophils pretreated with the anti-CD18 Ab, the EC
stiffness changed from 18.9 ± 0.8 dynes/cm2
to 20.8 ± 1.4, 20.6 ± 2.5, and 22.5 ± 2.9
dynes/cm2 (n = 3).
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for 8 or
24 h led to increases in ICAM-1 expression, whereas the expression
of E-selectin and P-selectin was not significantly altered (Table I). The roles of ICAM-1 in both the
stiffening response and the adhesion of neutrophils were thus examined
with anti-ICAM-1 Abs. As shown in Fig. 2a, 30 min pretreatment with
50 µg/ml murine anti-human ICAM-1 Ab (clone RR1/1), but not with
the control murine IgG, significantly attenuated EC stiffening response
induced by adherent neutrophils. Because the anti-ICAM-1 Ab had no
effect on neutrophil adherence (Fig. 2b), inhibition of the
stiffening response was not due to a decrease in the numbers of
neutrophils bound to the ECs. To confirm the lack of inhibition of
adhesion by this anti-ICAM-1 Ab, another clone of anti-ICAM-1
Ab (LB2) was used to pretreat ECs. This Ab also did not inhibit
neutrophil adherence to ECs (data not shown).
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, a and
b, treatment with 1% DMSO, a hydroxyl radical scavenger, or
0.3 mg/ml allopurinol, a xanthine oxidase inhibitor, inhibited the EC
stiffening response induced by adherent neutrophils. In addition,
treatment with 5 mM deferoxamine, an iron chelator, attenuated EC
stiffening response (Fig. 3c). These results suggest that
production of ROS is essential for inducing EC stiffening response. On
the other hand, treatment with 0.3 mM L-NMA
to inhibit production of nitric oxide had no effect, suggesting that
nitric oxide is not involved in this process (Fig. 3d).
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a, binding ICAM-1
with anti-ICAM-1 Ab (clone 6.5B5) did not alter the baseline
stiffness of 24-h TNF--treated ECs. However, cross-linking ICAM-1
with the rabbit anti-murine IgG Ab increased EC stiffness within 2
min. This increase persisted for at least 15 min. This
cross-linking-induced stiffening response required pretreatment with
anti-ICAM-1 Ab, as pretreatment with either mouse IgG or murine
anti-human HLA-A,B,C Ab had no effect. Cross-linking E-selectin
also did not alter the apparent stiffness of TNF--treated ECs (data
not shown), further supporting the specificity of the stiffening
response upon ICAM-1 ligation. The stiffening response also required
TNF- pretreatment, as ICAM-1 cross-linking did not increase the
apparent stiffness of untreated ECs (apparent stiffness of untreated
ECs before or 2, 6, 10, and 15 min after ICAM-1 cross-linking:
14.9 ± 0.3, 15.1 ± 0.8, 15.8 ± 1.1, 17.5 ± 1.3,
and 17.9 ± 1.0 dynes/cm2, respectively,
n = 4). This increase in the EC stiffness upon ICAM-1
cross-linking was inhibited by allopurinol (Fig. 4b),
suggesting that ROS are involved in the EC stiffening response upon
ICAM-1 cross-linking.
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The ROS required for EC stiffening may be derived from ROS
originating from neutrophils and/or ECs. To determine whether
neutrophils were the sources of ROS production during neutrophil-EC
adhesion, neutrophils were labeled with DCFDA and the oxidation of DCF
was measured. In neutrophils adherent to ECs for as long as 15 min,
little change in DCF fluorescence was observed whether neutrophils were
adherent to either untreated or TNF--treated ECs (Fig. 5a). To determine whether
neutrophils adherent to ECs are capable of producing oxidants, 1 µM
fMLP or buffer was added to neutrophils that had been adherent to
untreated ECs or to 24-h TNF--treated ECs for 15 min. Stimulation
with fMLP, but not buffer, induced a significant increase in DCF
fluorescence within 2 min, and this increase persisted for the 15 min
duration of the study (Fig. 5a). Together, these studies
suggest that neutrophils do not produce oxidants in response to
adherence to 24-hr TNF--treated ECs, although they can be stimulated
to produce oxidants by fMLP.
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-pretreated ECs
(3901 ± 393-unit increase during a 15-min interval vs 283 ± 15-unit
increase during this period (Fig. 5
b compared with Fig. 5a, p < 0.05). Treatment with 1 µM fMLP
resulted in further increases in oxidant production (Fig. 5b).
To determine whether the lack of oxidant production from neutrophils
bound to TNF--treated ECs compared with plastic wells or to
untreated ECs was due to decreased adhesion or adhesion-induced changes
in neutrophil shape, adhesion assays, and quantification of shape
changes were performed. Neutrophil adhesion assays demonstrated that
the percentage of neutrophils adherent to 24-h TNF--treated ECs at
15 min (16.8 ± 1.0%) was higher than that of neutrophils
adherent to uncoated plastic wells (7.3 ± 0.5%,
p < 0.05) or to untreated ECs (6.3 ± 0.6%,
p < 0.05). These data suggest that the lack of oxidant
production in neutrophils adherent to 24-hr TNF--treated ECs was not
due to less adhesion. Moreover, the lack of oxidant production was also
not due to lack of neutrophil shape changes. The projected area of
neutrophils adherent to 24-hr TNF--treated ECs (75.5 ± 7.0
µm2) was significantly larger than neutrophils
bound to untreated ECs (37.4 ± 2.2 µm2,
p < 0.05), and the measured increase in the ratio of
major axis length to the minor axis length indicated that neutrophils
adherent to 24-h TNF--treated ECs were more elongated than
neutrophils bound to untreated ECs (1.65 ± 0.08 vs 1.22 ±
0.02, p < 0.05). Together, these data suggest that
neutrophils adherent to 24-h TNF--treated ECs were well adhered,
spread, and elongated but did not make ROS until stimulated by
fMLP.
Oxidant production in ECs on neutrophil adherence: the roles of xanthine oxidase and ICAM-1
To determine whether ECs are the sources of ROS production, the
oxidant production in ECs was measured by labeling ECs with DCFDA. In
untreated ECs, the addition of neutrophils or buffer resulted in very
similar increases in the DCF fluorescence over 20 min (Fig. 6a). In contrast, in 24-h
TNF--treated ECs, addition of neutrophils induced a greater increase
in DCF fluorescence than addition of buffer (Fig. 6b). This
effect was apparent by 2 min and persisted for at least 20 min. These
data suggest that adherent neutrophils induced an increase in oxidant
production in 24-h TNF--treated, but not in untreated, ECs. This
increase in oxidant production was significantly attenuated by treating
ECs with 0.3 mg/ml allopurinol (Table II)
but not by treating ECs with 0.3 mM L-NMA (Table III), suggesting that activation of
xanthine oxidase was partially responsible for the oxidant production
in 24-h TNF--treated ECs upon neutrophil adherence.
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-treated ECs were treated with 50
µg/ml murine anti-human ICAM-1 Ab (clone RR1/1) for 30 min.
Treatment with this Ab, but not mouse IgG, inhibited the increase in EC
ROS production on neutrophil adherence (Table IV). These data suggest that
ICAM-1-mediated signaling events were involved in the oxidant
production in 24-h TNF--treated ECs on neutrophil adherence.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-activated human pulmonary microvascular ECs, but not to
untreated ECs, induced an increase in the apparent stiffness of ECs
within 2 min of adhesion. This stiffening response induced by
neutrophils was inhibited by an anti-ICAM-1 Ab and was mimicked by
cross-linking ICAM-1, suggesting that ICAM-1-mediated signaling events
may be involved. The neutrophil-induced EC stiffening response was
inhibited by DMSO, a hydroxyl radical scavenger, allopurinol, a
xanthine oxidase inhibitor, or deferoxamine, an iron chelator, but not
with L-NMA, a nitric oxide synthase inhibitor. These data
suggest that hydrogen peroxide and iron-dependent hydroxyl radicals
derived from xanthine oxidase-generated superoxide are involved in
mediating the EC stiffening response. Neutrophils adherent to 24-h
TNF--activated ECs produced almost nondetectable amounts of ROS,
although they responded to fMLP by generating ROS. However, neutrophil
adherence induced ROS production in 24-h TNF--activated ECs within 2
min, similar to the time required for the stiffening response. The
production of EC ROS was largely derived from a xanthine
oxidase-catalyzed reaction in ECs. The ICAM-1 Ab prevented the ROS
production in ECs. Taken together, on adherence of neutrophils, ICAM-1
may signal into ECs to produce ROS and initiate biomechanical changes
in ECs.
In this study, we used anti-ß1 integrin
Ab-coated beads to probe changes in the biomechanical properties of ECs
on neutrophil adherence. Previous studies have used beads coated with
synthetic RGD peptide or anti-ß1 integrin
Ab to evaluate the biomechanical properties of cultured ECs
(18). The results show similar apparent stiffness
measurements that are cytochalasin D-sensitive when either
type of beads was used, suggesting that ß1
integrin is an effective molecule for coupling the mechanical stress to
the cytoskeleton (18). In addition, the expression of
ß1 integrin on the EC surface was unaltered by
TNF- treatment, which allowed us to compare the apparent stiffness
measurement of untreated ECs and TNF--treated ECs without the
confounding effects of increased ß1 integrin
density on bead binding.
Neutrophil adherence induced changes in the biomechanical properties of
24-h TNF--treated pulmonary microvascular ECs that were ICAM-1
dependent. The ability of ICAM-1 to function as a signaling molecule
has been demonstrated in previous studies (14, 16, 17, 18, 19, 20). In
cultured ECs, ICAM-1 cross-linking induces increases in intracellular
Ca2+, activation of ErK-1 and AP-1 transcription
factor, as well as up-regulation of ICAM-1 and VCAM-1 mRNA (14, 17). Interestingly, these responses induced by ICAM-1
cross-linking mimic the responses induced by leukocyte adhesion
(17). Moreover, ICAM-1 activation by a mAb induces
activation of Rho, tyrosine kinase p60src, and
tyrosine phosphorylation of an actin cross-linking protein, cortactin,
in rat microvascular ECs (18, 19, 20). The interaction of
cortactin with F-actin is regulated by Src-mediated tyrosine
phosphorylation (21), and cortactin has been implicated in
the regulation of EC locomotion (22). Thus, neutrophil
adherence through ICAM-1 ligation induces signaling events in ECs,
which may ultimately modulate cytoskeleton organization.
Despite the inhibitory effects of the ICAM-1 Ab (clone RR1/1) on EC
stiffening response induced by neutrophils, this Ab had no effect on
neutrophil adherence to 24-h TNF--treated ECs. This is in contrast
to a previous report (26) in which the same
anti-ICAM-1 Ab partially inhibited neutrophil adherence to human
umbilical vein ECs stimulated with IL-1 for 4 h. Similar results
were obtained with another anti-ICAM-1 Ab (clone LB2). The basis
for this discrepancy remains to be determined. Neutrophil adherence to
24-h TNF--treated ECs required CD18, because an anti-CD18 Ab
completely inhibited neutrophil adhesion. Thus, neutrophil adhesion to
these cultured pulmonary microvascular ECs appears to be mediated
through CD11/CD18 binding to ligands other than ICAM-1. These possible
ligands include fibronectin, fibrinogen, and ICAM-2.
Our studies suggest that ICAM-1 signaling involves activation of xanthine oxidase and production of ROS. An anti-ICAM-1 Ab inhibited oxidant production in ECs as well as the EC stiffening response upon neutrophil adherence. In addition, the EC stiffening response induced by ICAM-1 cross-linking was inhibited by allopurinol. Activation of EC xanthine oxidase by neutrophils has been reported by several investigators. For instance, adherence of PMA-activated neutrophils to untreated ECs induces activation of xanthine oxidase in ECs, and neutrophil-derived hydrogen peroxide is not sufficient to induce this effect (23). In addition, leukotriene B4, thrombin, or PMA-induced neutrophil adherence to untreated ECs induces an increase in EC xanthine oxidase activity which is not inhibited by protease inhibitors and is not mimicked by exogenous hydrogen peroxide or neutrophil elastase (24). The importance of ICAM-1-induced signaling pathways in the activation of xanthine oxidase is supported by studies demonstrating that anti-ICAM-1 Ab completely blocks conversion of xanthine oxidase from the inactive xanthine dehydrogenase in ECs induced by neutrophil adherence in response to PMA (24, 25). These observations together with the studies presented in this paper suggest that activation of EC xanthine oxidase by adherent neutrophils may occur as the result of the signaling events induced by engagement of ICAM-1.
The inhibitory effect of allopurinol on the oxidant production was only partial, suggesting that other sources of superoxide may also contribute. Functional NADPH oxidase is expressed in cultured microvascular ECs (27), and membrane-associated NADPH oxidase activation in pulmonary endothelium is an important source of oxidants in lungs during ischemia injury (28). The roles of NADPH oxidase in mediating oxidant production in ECs after neutrophil adherence remain to be determined. Superoxide production by mitochondrial enzymes may also contribute. Our studies do exclude a role for nitric oxide in EC stiffening.
ROS generated at neutrophil adherence may modulate F-actin
organization. Recent evidence indicates that ROS may activate various
signal transduction pathways that are implicated in regulating F-actin
organization. Our previous studies demonstrated that neutrophil
adherence-induced increase in EC stiffness was
completely inhibited by treating ECs with
cytochalasin D, a F-actin disrupting agent, or jasplakinolide, an agent
that stabilizes F-actin, suggesting that F-actin rearrangement is
required for this stiffening response.5 Exogenous
hydrogen peroxide activates phospholipase D (29, 30, 31),
which has been shown to induce actin polymerization (32).
Moreover, hydrogen peroxide induces phosphatidylinositol
4,5-biphosphate formation in ECs (31), and
phosphatidylinositol 4,5-biphosphate regulates actin remodeling by
acting on several actin-binding proteins including gelsolin, vinculin,
profilin, and -actinin (33, 34, 35, 36). ROS also act as
intracellular second messengers and have been implicated in the
signaling cascades in response to growth factors and cytokines
(37, 38, 39). Thus, intracellular ROS, as second messengers,
may induce downstream signaling pathways that modulate the actin
cytoskeleton. How these signaling events are transduced in ECs on
neutrophil adherence is unclear. Changes in DCF fluorescence allow us
to assess only the gross changes in oxidant production in the cells.
Other investigators have postulated that the generation of oxidants
inside the cells may be highly localized, and the site of production
may be critical for the effect of oxidants on signaling pathways
(37, 38, 39).
Although the ROS mediating EC stiffening response could be formed from
neutrophil-derived ROS and/or from EC-derived ROS, this study clearly
shows that ROS generated during neutrophil adhesion to
TNF--activated ECs were not derived from neutrophils. In contrast,
neutrophils adherent to untreated ECs or uncoated plastic wells did
produce DCF-oxidizing ROS in response to fMLP stimulation (Fig. 5a compared with Fig. 5b). This lack of ROS
production was not due to less adherence, because the percentage and
the number of neutrophils adherent to 24-h TNF--activated ECs were
higher than those adherent to the uncoated plastic wells. In addition,
this difference was not due to global inhibition of neutrophil
activation by TNF--activated ECs, because fMLP induced radical
production in both EC- and plastic-adherent neutrophils. Finally, the
lack of ROS was not due to lack of neutrophil shape changes or
cytoskeletal rearrangements, because neutrophils became spread and
elongated when adherent to TNF--activated ECs but not untreated ECs.
Thus, neutrophil spreading and F-actin reorganization upon adherence to
24-h TNF--activated ECs are not sufficient to generate ROS in
neutrophils.
This study clearly demonstrates that neutrophil adhesion to ECs induces signaling pathways in ECs through ligation of ICAM-1 that result in stiffening of these cells. The function of this stiffening, as well as its molecular mechanisms, is not yet clear. One possibility is that it represents the first phase of neutrophil-induced EC injury and death. However, a more likely possibility is that changes in the actin cytoskeleton modulate EC shape and surface characteristics that facilitate neutrophil migration toward the EC junctions. Cytoskeletal changes may also alter the functions of paracellular junctions. Recent studies from our laboratory have demonstrated that inhibiting this cytoskeletal stiffening does in fact prevent neutrophil migration to the borders of ECs. Finally, cytoskeletal reorganization may regulate signaling pathways by modulating their cytoplasmic compartmentalization. These changes in ECs induced by adherent neutrophils may contribute to EC permeability changes and modulate neutrophil emigration through the EC junctions in inflammation.
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2 Current address: Rainbow Babies and Children’s Hospital, Room 8321, 11100 Euclid Ave., Cleveland, OH 44106.
3 Address correspondence and reprint requests to Claire M. Doerschuk, M.D., Rainbow Babies and Children’s Hospital, Room 8321, 11100 Euclid Ave., Cleveland, OH 44106.
4 Abbreviations used in this paper: ECs, endothelial cells; L-NMA, N-methyl-L-arginine; ROS, reactive oxygen species; DCF, dichlorofluorescein; DCFDA, 2',7'-dichlorofluorescein diacetate.
5 Q. Wang, E. T. Chiang, M. Lim, J. Lai, R. Rogers, P. A. Janmey, D. Shepro, and C. M. Doerschuk. Changes in the biomechanical properties of neutrophils and endothelial cells during adhesion. Submitted for publication.
Received for publication October 15, 1999. Accepted for publication March 23, 2000.
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B-independent mechanism. J. Immunol. 162:2990.
1 by profilin and tyrosine phosphorylation. Science 251:1231.-actinin. J. Biol. Chem. 271:2646.This article has been cited by other articles:
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|
  R. Sumagin, E. Lomakina, and I. H. Sarelius Leukocyte-endothelial cell interactions are linked to vascular permeability via ICAM-1-mediated signaling Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H969 - H977. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Hecquet, G. U. Ahmmed, S. M. Vogel, and A. B. Malik Role of TRPM2 Channel in Mediating H2O2-Induced Ca2+ Entry and Endothelial Hyperpermeability Circ. Res., February 15, 2008; 102(3): 347 - 355. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Sumagin and I. H. Sarelius A role for ICAM-1 in maintenance of leukocyte-endothelial cell rolling interactions in inflamed arterioles Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H2786 - H2798. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Wu, R. Guo, Y. Wang, and P. N. Cunningham The role of ICAM-1 in endotoxin-induced acute renal failure Am J Physiol Renal Physiol, October 1, 2007; 293(4): F1262 - F1271. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Urquhart, G. Rosignoli, D. Cooper, R. Motterlini, and M. Perretti Carbon Monoxide-Releasing Molecules Modulate Leukocyte-Endothelial Interactions under Flow J. Pharmacol. Exp. Ther., May 1, 2007; 321(2): 656 - 662. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Yang, R. M. Froio, T. E. Sciuto, A. M. Dvorak, R. Alon, and F. W. Luscinskas ICAM-1 regulates neutrophil adhesion and transcellular migration of TNF-{alpha}-activated vascular endothelium under flow Blood, July 15, 2005; 106(2): 584 - 592. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Wang, M. Yerukhimovich, W. A. Gaarde, I. J. Popoff, and C. M. Doerschuk MKK3 and -6-dependent activation of p38{alpha} MAP kinase is required for cytoskeletal changes in pulmonary microvascular endothelial cells induced by ICAM-1 ligation Am J Physiol Lung Cell Mol Physiol, February 1, 2005; 288(2): L359 - L369. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Y. Davani, D. R. Dorscheid, C.-H. Lee, C. van Breemen, and K. R. Walley Novel regulatory mechanism of cardiomyocyte contractility involving ICAM-1 and the cytoskeleton Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1013 - H1022. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Alvarez-Maqueda, R. E. Bekay, G. Alba, J. Monteseirin, P. Chacon, A. Vega, J. Martin-Nieto, F. J. Bedoya, E. Pintado, and F. Sobrino 15-Deoxy-{Delta}12,14-prostaglandin J2 Induces Heme Oxygenase-1 Gene Expression in a Reactive Oxygen Species-dependent Manner in Human Lymphocytes J. Biol. Chem., May 21, 2004; 279(21): 21929 - 21937. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. van Buul and P. L. Hordijk Signaling in Leukocyte Transendothelial Migration Arterioscler Thromb Vasc Biol, May 1, 2004; 24(5): 824 - 833. [Abstract] [Full Text]
|
|
|
|
|
|
Q. Wang, G. R. Pfeiffer II, and W. A. Gaarde Activation of SRC Tyrosine Kinases in Response to ICAM-1 Ligation in Pulmonary Microvascular Endothelial Cells J. Biol. Chem., November 28, 2003; 278(48): 47731 - 47743. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Kingham, W. G. McLean, M.-T. Walsh, A. D. Fryer, G. J. Gleich, and R. W. Costello Effects of eosinophils on nerve cell morphology and development: the role of reactive oxygen species and p38 MAP kinase Am J Physiol Lung Cell Mol Physiol, October 1, 2003; 285(4): L915 - L924. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Burns, C. W. Smith, and D. C. Walker Unique Structural Features That Influence Neutrophil Emigration Into the Lung Physiol Rev, April 1, 2003; 83(2): 309 - 336. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. P. Bowler, J. Arcaroli, E. Abraham, M. Patel, L.-Y. Chang, and J. D. Crapo Evidence for extracellular superoxide dismutase as a mediator of hemorrhage-induced lung injury Am J Physiol Lung Cell Mol Physiol, April 1, 2003; 284(4): L680 - L687. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Wang, G. R. Pfeiffer II, T. Stevens, and C. M. Doerschuk Lung Microvascular and Arterial Endothelial Cells Differ in Their Responses to Intercellular Adhesion Molecule-1 Ligation Am. J. Respir. Crit. Care Med., September 15, 2002; 166(6): 872 - 877. [Abstract] [Full Text]
|
|
|
|
 |
|
 G. Ji, C. D. O'Brien, M. Feldman, Y. Manevich, P. Lim, J. Sun, S. M. Albelda, and M. I. Kotlikoff PECAM-1 (CD31) regulates a hydrogen peroxide-activated nonselective cation channel in endothelial cells J. Cell Biol., April 1, 2002; 157(1): 173 - 184. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Saito, J. Lai, R. Rogers, and C. M. Doerschuk Mechanical properties of rat bone marrow and circulating neutrophils and their responses to inflammatory mediators Blood, March 15, 2002; 99(6): 2207 - 2213. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X.-p. Gao, N. Xu, M. Sekosan, D. Mehta, S. Y. Ma, A. Rahman, and A. B. Malik Differential Role of CD18 Integrins in Mediating Lung Neutrophil Sequestration and Increased Microvascular Permeability Induced by Escherichia coli in Mice J. Immunol., September 1, 2001; 167(5): 2895 - 2901. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. P. BOWLER, J. ARCAROLI, J. D. CRAPO, A. ROSS, J. W. SLOT, and E. ABRAHAM Extracellular Superoxide Dismutase Attenuates Lung Injury after Hemorrhage Am. J. Respir. Crit. Care Med., July 15, 2001; 164(2): 290 - 294. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Wang and C. M. Doerschuk The p38 Mitogen-Activated Protein Kinase Mediates Cytoskeletal Remodeling in Pulmonary Microvascular Endothelial Cells Upon Intracellular Adhesion Molecule-1 Ligation J. Immunol., June 1, 2001; 166(11): 6877 - 6884. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Wang, E. T. Chiang, M. Lim, J. Lai, R. Rogers, P. A. Janmey, D. Shepro, and C. M. Doerschuk Changes in the biomechanical properties of neutrophils and endothelial cells during adhesion Blood, February 1, 2001; 97(3): 660 - 668. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Ji, C. D. O'Brien, M. Feldman, Y. Manevich, P. Lim, J. Sun, S. M. Albelda, and M. I. Kotlikoff PECAM-1 (CD31) regulates a hydrogen peroxide-activated nonselective cation channel in endothelial cells J. Cell Biol., April 1, 2002; 157(1): 173 - 184. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Kingham, W. G. McLean, D. A. Sawatzky, M. T. Walsh, and R. W. Costello Adhesion-dependent interactions between eosinophils and cholinergic nerves Am J Physiol Lung Cell Mol Physiol, June 1, 2002; 282(6): L1229 - L1238. [Abstract] [Full Text] [PDF]
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) was measured by magnetic twisting cytometry using ferromagnetic
beads bound to ß1 integrin as described in
Materials and Methods. After the measurement of
stiffness baseline, purified neutrophils (neutrophil:EC, 1:1) were
added to the well, and the EC stiffness was measured 2, 6, 10, and 15
min later. Data are expressed as means ± SEM
(n = 4). *, p < 0.05
compared with the baseline stiffness.
) or control murine IgG (
, 
, Neutrophils
adherent to 24-h TNF-