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Mediation of endothelial cell damage by serine proteases but not superoxide released from antineutrophil cytoplasmic antibodystimulated neutrophils.

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Vol. 54, No. 5, May 2006, pp 1619–1628
DOI 10.1002/art.21773
© 2006, American College of Rheumatology
Mediation of Endothelial Cell Damage by
Serine Proteases, but Not Superoxide, Released From
Antineutrophil Cytoplasmic Antibody–Stimulated Neutrophils
X. Lu, A. Garfield, G. E. Rainger, C. O. S. Savage, and G. B. Nash
Conclusion. Endothelial cells inhibit superoxide
generation by fMLP and ANCA-activated neutrophils.
The release of vWF occurs during coculture and is
sensitive to serine protease, but not NADPH oxidase
inhibition. Serine proteases may play a more important
role than reactive oxygen species as mediators of endothelial injury during ANCA-associated systemic vasculitis.
The vascular endothelium fulfills a number of
important functions in the maintenance of the circulation. These functions include prevention of coagulation,
regulation of vascular tone and permeability, and control of leukocyte recruitment. Both the innate and
adaptive immune responses are dependent on the migration of leukocytes across endothelial cells. Inflammatory stimuli activate the microvascular endothelial cells
to express adhesion molecules (including members of
the selectin family) and chemoattractant compounds
that physically engage circulating leukocytes and promote their adhesion and migration. Injury or altered
responses of endothelial cells during pathogenic processes could grossly alter these functions and prevent
resolution of inflammatory responses.
The endothelium is injured during small-vessel
systemic vasculitis, particularly the triad of diseases
composed of Wegener’s granulomatosis, microscopic
polyangiitis, and Churg-Strauss syndrome, which are
associated with the presence of antineutrophil cytoplasmic autoantibodies (ANCAs). Early in the vasculitic
process, endothelial cells become swollen and denuded
from the basement membrane (1). Increased numbers of
endothelial cells are present within the circulation during active vasculitis (2). As the process progresses,
inflammatory cells invade and surround the vessel wall,
and areas of fibrinoid necrosis develop within the wall
(3). The extent to which endothelial cells are passive
recipients of injury inflicted by activated leukocytes or to
Objective. To evaluate potential mediators of endothelial cell injury in systemic vasculitis associated
with antineutrophil cytoplasmic antibodies (ANCAs),
we investigated the factors controlling the neutrophil
respiratory burst and endothelial release of von Willebrand factor (vWF) during neutrophil–endothelial cell
Methods. Superoxide release from neutrophils
binding to purified P-selectin or to tumor necrosis
factor–activated endothelial cells was measured under
flow or static conditions using the superoxide dismutase
(SOD)–inhibitable reduction of ferricytochrome c. Neutrophils were activated with fMLP, normal IgG, or
ANCA IgG. Enzyme-linked immunosorbent assay was
used to measure vWF. Serine protease activity was
measured enzymatically.
Results. ANCA IgG or fMLP induced superoxide
release when perfused over neutrophils that were rolling
over P-selectin, but not those that were binding to
endothelial cells. In static assays, endothelial cells inhibited superoxide production by neutrophils. Adenosine inhibited the respiratory burst, and, in cocultures,
adenosine deaminase overcame the inhibitory effects of
endothelial cells. Serine proteases were released during
activated neutrophil–endothelial cell coculture. There
was enhanced release of vWF during activated
neutrophil–endothelial cell coculture; this was not inhibited by diphenyleneiodonium or by SOD plus catalase, but was inhibited by diisopropylfluorophosphate.
Supported by the Arthritis Research Campaign.
X. Lu, PhD, A. Garfield, PhD, G. E. Rainger, PhD, C. O. S.
Savage, MD, PhD, FMedSci, G. B. Nash, PhD: University of Birmingham, Birmingham, UK.
Address correspondence and reprint requests to C. O. S.
Savage, MD, PhD, FMedSci, Division of Immunity and Infection, The
School of Medicine, University of Birmingham, Birmingham B15 2TT,
UK. E-mail:
Submitted for publication August 16, 2005; accepted in
revised form January 19, 2006.
which they promote the development of vascular injury
remains a matter of conjecture.
A popular hypothesis is that endothelial injury
derives from the binding of ANCAs to neutrophils and
the subsequent disturbance of neutrophil–endothelial
interactions. ANCAs are IgG autoantibodies that are
directed against the neutrophil granule components
proteinase 3 (PR3), a serine protease that cleaves a
variety of proteins, and myeloperoxidase (MPO), a
heme-containing peroxidase that uses chloride to generate hypochlorous acid and other reactive oxidants from
hydrogen peroxide. Animal models have demonstrated
that infusion of ANCA IgG is sufficient to induce
vasculitic lesions in mice (4). In vitro, ANCA IgG
activate a variety of neutrophil functions, inducing the
release of superoxide (5), degranulation (6), and the
release of interleukin-8 (IL-8) (7) and IL-1 (8). They
induce neutrophils to become firmly adherent to endothelial cells, following which increased numbers of neutrophils migrate across the endothelial barrier (9).
It is the ability of ANCA IgG to combine neutrophil activation and release of potentially cytotoxic
agents with enhanced adhesion to endothelium that has
suggested links between the autoantibodies, neutrophils,
and the vascular damage. Indeed, in vitro assays of
endothelial damage have supported the notion that
ANCA-activated neutrophils promote endothelial injury
(10). However, questions arise as to whether neutrophils
normally release superoxide and modify endothelial
function during their adhesion and migration through
endothelial monolayers or whether ANCAs or other
systemic activating agents (such as the bacterial peptide
fMLP) might cause such potentially dangerous responses.
To address these questions, we used in vitro flow
models to test responses of neutrophils to activation by
ANCA IgG or fMLP, either as they “rolled” on the
purified adhesion receptor P-selectin or as they adhered
and migrated on monolayers of endothelial cells activated with the cytokine tumor necrosis factor ␣ (TNF␣).
During the course of these studies, we noticed that
superoxide release from fMLP- or ANCA-stimulated
neutrophils adherent on P-selectin was greater than that
from similar neutrophils adherent on endothelial cells.
Examining this further using static coculture assays, we
demonstrated that endothelial cells suppressed neutrophil superoxide production by releasing adenosine. Nevertheless, endothelial cells cocultured with fMLP- or
ANCA-activated neutrophils released von Willebrand
factor (vWF), a phenomenon previously used as an
indicator of endothelial damage in vasculitis. The release of vWF was sensitive to inhibition by serine
protease inhibitors, but not to inhibition by NADPH
oxidase inhibitors. Thus, while activation of adherent
neutrophils can induce oxidant production, this is suppressed by endothelial cells, which are nevertheless
acted upon by a protease(s) released by neutrophils
when they are stimulated by exogenous activators.
Isolation of human polymorphonuclear neutrophils.
Blood from healthy volunteers was collected into tubes containing potassium EDTA (Sarstedt, Leicester, UK). Neutrophils were isolated by centrifuging the whole blood at 800g for
30 minutes over a 2-step density gradient consisting of equal
quantities of Histopaques 1119 and 1077 (Sigma, Poole, UK)
as described previously (11). The neutrophils were washed
twice in phosphate buffered saline (PBS) containing 1 mM
calcium and 0.5 mM magnesium (Gibco BRL, Paisley, UK)
and 0.1% bovine serum albumin (BSA) (fraction IV; Sigma),
counted using a Coulter counter (Coulter Electronics, Luton,
UK), and adjusted to 106/ml in the same medium.
Preparation of immunoglobulins. Human IgG was
isolated from patients with ANCA-associated vasculitis (PR3
ANCA or MPO ANCA), as well as from healthy controls by
affinity chromatography using a HiTrap protein G affinity
column (Pharmacia, Uppsala, Sweden) and pyrogen-free materials, as previously described (8). The specificity of anti-PR3
or anti-MPO ANCA IgG was assessed by antigen-specific
enzyme-linked immunosorbent assay (ELISA).
Coating of microslides with purified P-selectin. Microslides (glass capillary tubes with a rectangular cross section of
3 ⫻ 0.3 mm and a length of 50 mm; Camlab, Cambridge, UK)
were acid-washed and treated with 3-aminopropyltriethoxysilane (APES) (Sigma) as described previously (12).
Purified P-selectin (2 ␮g/ml) in PBS was aspirated into the
microslides and incubated at 37°C for 60 minutes to allow
binding to the APES. The microslides were then washed and
filled with 1% BSA (fraction IV) in PBS and incubated at 37°C
for a further 60 minutes to block any free protein-binding sites.
Endothelial cell culture. Human umbilical vein endothelial cells (HUVECs) were isolated as described previously
(12) and cultured in medium 199 (Gibco BRL) containing 20%
fetal calf serum (Sigma) and 10 ng/ml of endothelial cell
growth factor (Sigma). Confluent primary cultures were dispersed using trypsin/EDTA (Sigma) and were seeded into and
cultured in APES-treated microslides as described previously
(12,13). Seeding was at a level previously determined to give a
confluent monolayer following 24 hours of culture. Alternatively, HUVECs were seeded into 96-well flat-bottomed microtiter plates at a concentration of 5 ⫻ 104/well and allowed
to grow to confluence over 24 hours. In either case, confluent
HUVECs were stimulated with 0, 2, 5, or 100 units/ml of TNF␣
for 4 hours prior to assays. Each experiment was conducted
with HUVECs from a different primary culture from a single
Adhesion and superoxide release by neutrophils under
conditions of flow. The assay we used was based on the
previously described assay (14). A microslide containing adhesive substrate (P-selectin or TNF-treated endothelial cells) was
attached to the common outlet of a 4-port tap (Hamilton
miniature valve; VA Howe, London, UK). Plastic 2.5-ml
perfusion syringes containing either activating agent, PBS–
BSA, or a suspension of neutrophils were connected to 1 of the
3 remaining tap inlets via flexible silicone tubing. These
syringes were mounted back-to-back with identical water-filled
slave syringes on a specially engineered rig that held them
immobile. Expulsion of the perfusate was driven at a controlled rate by a 50-ml master syringe on a Harvard syringe
pump that pushed flow into the slave syringes. Selection
between perfusates for the microslide was achieved using 2
electronic valves (Lee Products, Gerrards Cross, UK) that
switched the route of the output from the master syringe to a
chosen slave syringe. A constant wall shear stress of 0.1 Pa (or
1 dyne/cm2) was maintained in the microslide by maintaining
the flow rate at 0.382 ml/minute.
Microslides were mounted on the stage of a microscope fitted with a video camera, monitor, and recorder. The
whole perfusion system was maintained at 37°C. Neutrophils
were perfused across adhesive substrates at a concentration of
106/ml for 2.5 minutes. Perfused cells that were close to the
wall of the microslide adhered via P-selectin and formed
rolling attachments. Neutrophils adhering to TNF-treated
endothelial cells either rolled slowly over the surface, became
immobilized and migrated on the surface, or went on to
migrate through the endothelial monolayer (13). Nonadherent
neutrophils were removed from the microslide with a 2-minute
perfusion of PBS–BSA, leaving a large population of attached
neutrophils in either case. A video record was made in at least
5 fields of view of known dimensions to allow the number of
adherent cells to be counted.
After washout, the perfusate was switched so that
adherent neutrophils were treated with activating compounds,
either ANCA IgG (200 ␮g/ml), normal IgG (200 ␮g/ml), or
fMLP (1 ␮M). To enable simultaneous measurement of superoxide release, the PBS–BSA also contained 75 ␮M ferricytochrome c (Sigma), with or without 300 units/ml of superoxide
dismutase (SOD). The stimulus/ferricytochrome c solution was
perfused continuously for 10 minutes, and the perfusate was
collected from the outlet in 1-minute fractions for assay of
superoxide production. The aliquots were centrifuged at
13,000 revolutions per minute for 1 minute to clear them, and
absorbance was measured at 550 nm. Superoxide production
was calculated as previously described (5) and expressed as
nanomoles per 105 adherent cells. The peak superoxide release
usually occurred after 5 minutes, and the maximum release was
calculated from the mean superoxide release from the 3
samples taken from immediately before, at the time of, and
immediately after the peak release.
Measurement of neutrophil superoxide production on
endothelial cells in multiwell plates. Plates containing endothelial cells were washed 3 times with warmed PBS. Reaction
buffers (200 ␮l) containing 75 ␮M ferricytochrome c in PBS,
with either 300 units/ml of SOD or an equal volume of PBS,
were added. Then, primed neutrophils (105/50 ␮l) were placed
in contact with endothelial cell monolayers or in wells without
endothelial cell monolayers. Prior to addition of neutrophils to
the wells, priming was performed for 15 minutes at 37°C using
TNF␣ (80 units/ml) and cytochalasin B (5 ␮g/ml). Once in the
wells, the neutrophils were stimulated with 200 ␮g/ml of
ANCA IgG, 200 ␮g/ml of normal IgG, or 1 ␮M fMLP.
Superoxide production was determined continuously at 37°C
using a kinetic microplate assay (5). In some experiments,
adenosine (Sigma) at a concentration of 1 ␮M, 10 ␮M, 100 ␮M,
or 500 ␮M or the nonselective adenosine receptor antagonist
8-(p-sulfophenyl)-theophylline (8-SPT; Sigma) at concentrations between 1 ␮M and 1,000 ␮M was added to neutrophils in
the absence of endothelial cells. In other experiments, adenosine deaminase (Sigma) at concentrations between 0.1 units/ml
and 1.0 units/ml was added to neutrophils in the presence of
endothelial cells.
Measurement of the release of vWF by endothelial
cells. Endothelial cell monolayers in 96-well tissue culture
plates were rinsed 3 times with warm PBS, followed by the
addition of 105 neutrophils in 50 ␮l and 200 ␮l of reaction
buffer alone or in reaction buffer containing either SOD (150
units/ml), which converts superoxide to hydrogen peroxide,
diphenyleneiodonium (DPI; 5 mM), which inhibits the formation of reactive oxygen species by the NADPH oxidase complex, or diisopropylfluorophosphate (DFP; 1 mM, 2 mM, or 5
mM), a noncompetitive irreversible inhibitor of serine proteases. The mixture was incubated for 5 minutes at 37°C, then
fMLP (1 ␮M), normal IgG (200 ␮g/ml), or ANCA IgG (200
␮g/ml) was added, and incubation was continued for 3 hours at
37°C. Supernatants were collected for vWF measurements.
Levels of vWF antigen were determined by sandwich
ELISA. Briefly, 96-well ELISA plates were coated with 100 ␮l
(in each well) of a 1:500 dilution of rabbit anti-human vWF in
carbonate buffer (Dako A082; Dako, Glostrup, Denmark), and
the plates were incubated for 1 hour at room temperature. The
coated plate was washed 3 times with PBS–0.05% Tween
(PBST). Sample supernatants (100 ␮l each) were applied to
plates in duplicate and incubated for 1 hour at room temperature. The plate was washed 3 times with PBST and incubated
with 100 ␮l of horseradish peroxidase–conjugated rabbit antihuman vWF (diluted 1:2,000 in PBST) for 1 hour at room
temperature. After washing with PBST, a peroxidase reaction
was performed by adding 100 ␮l of PBS containing 0.66%
(weight/volume) o-phenylenediamine (Dako S2045) and hydrogen peroxide, and incubating in the dark for 5 minutes at
room temperature.
The absorbance at 495 nm was measured with a plate
reader after the addition of 100 ␮l of 2M sulfuric acid. The
vWF concentration was expressed in international units per
deciliter, which was standardized with the use of a reference
vWF from the National Institute for Biological Standards and
Controls. The assay was originally set up with a standard curve
appropriate for measuring vWF in plasma; the standard curve
was nonlinear, and when the assay was applied to tissue culture
supernatants, some values that fell below the standard curve
gave negative values. To account for this, values obtained with
untreated endothelial cells cultured in the absence of neutrophils (which routinely gave negative values) were subtracted
from treated samples. In some experiments, endothelial cells
were treated with PR3 (Athens Research and Technology,
Athens, GA) at concentrations of 1–10 ␮g/ml for 180 minutes
at 37°C, and release of vWF into supernatants was measured as
described above.
Measurement of neutrophil elastase and PR3 release
from the coculture system. The release of the serine proteases
elastase and PR3 into culture medium was assayed using 100 ␮l
of the synthetic common substrate (2 mM) N-methoxysuccinylAla-Ala-Pro-Val p-nitroanilide in 0.1% Tween 20 and 1.25%
DMSO. This was mixed with 100 ␮l of coculture supernatant
and incubated for 22 hours at 37°C. The color development
was monitored at 405 nm with a Titertek Multiscan (Titertek
Flow, Zwanenburg, The Netherlands).
Statistical analysis. Effects of time and treatment were
tested by analysis of variance using Microsoft Excel software
(Microsoft, Redmond, WA). Comparisons between 2 individual treatments were done using paired or unpaired t-tests as
Superoxide release from neutrophils adherent to
P-selectin or endothelial cells in a flow system. Neutrophils perfused over P-selectin at a wall shear stress of 0.1
Pa bound efficiently (⬃2,000/mm2), with ⬎97% of the
adherent cells rolling. When fMLP (1 ␮M) was perfused
over the rolling cells, all neutrophils rapidly became
stationary adherent and changed shape, and they migrated over the surface as described previously (15).
After perfusion of ANCA IgG (200 ␮g/ml), their behavior changed more slowly. After 5 minutes, the number of
cells in contact with the P-selectin–coated microslide
was unaltered, but the proportion of rolling cells had
fallen to 62%. After 10 minutes of perfusion, the majority of neutrophils in contact with the P-selectin–coated
microslide were firmly adherent and stationary, and the
percentage of rolling cells was 48%. The shape of the
cells changed from spherical to irregular with pseudopodia. Normal IgG did not induce such changes. Similar
responses have been described in a previous report (16).
When ANCA IgG or fMLP was superfused over
neutrophils that were rolling on P-selectin, there was a
Figure 1. Superoxide release by neutrophils binding to P-selectin in
the flow-based assay. Rolling neutrophils were untreated or were
superfused with normal IgG (200 ␮g/ml), antineutrophil cytoplasmic
antibody (ANCA) IgG (200 ␮g/ml), or fMLP (1 ␮M). Values are the
mean and SEM of 5–8 experiments. Analysis of variance showed a
significant effect of treatment (P ⬍ 0.01). ⴱ ⫽ P ⬍ 0.05; ⴱⴱ⫽ P ⬍ 0.01
by paired t-test.
Figure 2. Effect of endothelial cells on the time course of superoxide
release by neutrophils stimulated with A, fMLP (1 ␮M) or B, antineutrophil cytoplasmic antibody (ANCA) IgG (200 ␮g/ml). Neutrophils
(105) were added to wells without endothelial cells (EC) or to wells
with endothelial cells treated with the indicated concentrations of
tumor necrosis factor ␣ (TNF␣) for 4 hours. Values are the mean ⫾
SEM of 4 experiments in A and 3 experiments in B. Analysis of
variance (ANOVA) showed a significant effect of time and presence of
endothelial cells on superoxide release in the experiments shown in
both A and B (P ⬍ 0.01 in each case). In addition, in A, ANOVA
showed a significant effect of the TNF␣ dose when endothelial cells
were present (P ⬍ 0.01).
significant release of superoxide, as detected by continuous conversion of ferricytochrome c and color change
of the perfusate (Figure 1). Superfusion of normal IgG
or PBS–BSA did not induce detectable release of superoxide (Figure 1).
Neutrophils perfused over TNF-treated endothelial cells also formed numerous attachments with a
mixture of adhesive behaviors. When either fMLP or
ANCA was perfused over these cells, rolling ceased and
essentially all cells were stationary adherent. However,
superoxide production from neutrophils was not detectable in the presence of an endothelial cell monolayer
within the microslide, even though an equivalent number of neutrophils bound as with P-selectin (data not
Figure 3. A, Effects of various concentrations of adenosine on superoxide release by neutrophils treated with fMLP (1 ␮M) or antineutrophil
cytoplasmic antibody (ANCA) IgG (200 ␮g/ml) in the absence of endothelial cells. Each well contained 105 neutrophils, and superoxide was
measured after 180 minutes. Analysis of variance (ANOVA) showed a significant effect of treatment on superoxide release induced by fMLP or
ANCA IgG (P ⬍ 0.01 in each case). B, Dose response for the effect of adenosine deaminase on the release of superoxide by neutrophils incubated
with endothelial cells (EC) and treated with fMLP (1 ␮M). Neutrophils (105) were added to each well with or without endothelial cells. ANOVA
showed a significant effect of adenosine deaminase treatment on superoxide release (P ⬍ 0.01). C, Effects of adenosine deaminase on superoxide
release by neutrophils incubated with endothelial cells and treated with ANCA or fMLP. Neutrophils (105) were untreated or were treated with
normal IgG (200 ␮g/ml), ANCA IgG (200 ␮g/ml), or fMLP (1 ␮M) and were added to wells with or without unstimulated endothelial cells and in
the presence or absence of adenosine deaminase (ADA; 0.5 units/ml). Superoxide was measured after 180 minutes of coculture. ANOVA showed
that treatments of neutrophils or of endothelial cells had significant effects (P ⬍ 0.01 in each case). ⴱⴱ ⫽ P ⬍ 0.01 versus no endothelial cells; ⫹
⫽ P ⬍ 0.05 and ⫹⫹ ⫽ P ⬍ 0.01 versus endothelial cells alone, by paired t-test. Values in A–C are the mean and SEM of 3 experiments.
shown). Thus, while activated neutrophils released superoxide when adhered to a surface coated with
P-selectin and albumin, this response was inhibited in
the presence of endothelial cells.
Inhibition of fMLP- or ANCA IgG–stimulated
neutrophil superoxide production by endothelial cells.
To examine further the ability of endothelial cells to
inhibit superoxide release, neutrophils were settled on
monolayers that had been pretreated with different
concentrations of TNF␣ and were then stimulated with
fMLP or ANCA IgG. The presence of an endothelial
cell monolayer continuously reduced the release of
superoxide from neutrophils that had been stimulated
with fMLP over 180 minutes (Figure 2A). The inhibitory
effect was strongest for unstimulated endothelial cells
and was partly abrogated if the endothelial cells were
activated with increasing concentrations of TNF␣. When
neutrophils were stimulated with ANCA IgG, a similar,
prolonged inhibitory effect was seen in the presence of
endothelial cells that had been pretreated with TNF␣ (2
units/ml) for 4 hours (Figure 2B). Normal IgG induced
minimal release of superoxide in the presence or absence of endothelial cells (data not shown).
Inhibition of neutrophil superoxide production
by endothelial cell–derived adenosine. The mediator of
the inhibitory effects of endothelial cells on neutrophil
superoxide production was then considered. In other
systems, adenosine has been shown to mediate such
effects (17). Initially, we determined whether adenosine
could inhibit neutrophil superoxide production by adding it to neutrophils in blank wells. Increasing concentrations of adenosine inhibited superoxide production
from fMLP- and ANCA IgG–stimulated neutrophils in a
dose-dependent manner (Figure 3A). The inhibitory
effects of adenosine (at 100 ␮M) could be attenuated by
the adenosine A1 and A2 nonselective receptor antagonist 8-SPT in a dose-dependent manner at concentrations ranging from 1 ␮M to 1,000 ␮M (data not shown).
To determine whether adenosine was the mediator of the inhibitory effects in the coculture systems,
adenosine deaminase (which breaks down adenosine)
was added. Increasing concentrations of adenosine
deaminase led to a restoration of superoxide production
from fMLP-stimulated neutrophils cultured in the presence of endothelial cells (Figure 3B). In further experiments, the effects of adenosine deaminase at 0.5 units/ml
Figure 4. Release of von Willebrand factor (vWF) from endothelial
cells cultured with neutrophils. Neutrophils (105) were untreated or
were treated with normal IgG (200 ␮g/ml), antineutrophil cytoplasmic
antibody (ANCA) IgG (200 ␮g/ml), or fMLP (1 ␮M), and vWF was
measured after 180 minutes of coculture. Values are the mean and
SEM of 3–5 experiments. Analysis of variance showed a significant
effect of neutrophil treatment (P ⬍ 0.01). ⴱ ⫽ P ⬍ 0.05 versus normal
IgG; ⫹⫹ ⫽ P ⬍ 0.01 versus untreated, by paired t-test.
Figure 5. Protease release from neutrophils cultured with endothelial
cells. Neutrophils (105) were untreated or were treated with normal
IgG (200 ␮g/ml), antineutrophil cytoplasmic antibody (ANCA) IgG
(200 ␮g/ml), or fMLP (1 ␮M). Protease activity (absorbance from
substrate digestion assay) was measured after 180 minutes of coculture. Values are the mean and SEM of 3–5 experiments. Analysis of
variance showed a significant effect of neutrophil treatment (P ⬍ 0.01).
ⴱ ⫽ P ⬍ 0.05 versus normal IgG; ⫹⫹ ⫽ P ⬍ 0.01 versus untreated, by
Student’s t-test.
were compared for neutrophil superoxide responses to
stimulation with ANCA IgG, normal IgG, or fMLP in
the presence of endothelial cells. In each case, adenosine
deaminase was able to overcome the inhibitory effects of
endothelial cells on neutrophil superoxide production
(Figure 3C).
It seemed likely that endothelial cells were the
direct source of adenosine production in the coculture
system, although it was possible that they carried out
extracellular phosphohydrolysis of adenine nucleotides
from neutrophils via surface ectonucleotidases such as
CD39 or CD73. To examine this further, we harvested
supernatants from resting endothelial cells and added
these to 105 fMLP-stimulated neutrophils in the presence or absence of adenosine deaminase. Superoxide
production was 13.4 ⫾ 0.3 nmoles (mean ⫾ SEM) from
fMLP-stimulated neutrophils, 6.1 ⫾ 0.7 nmoles in the
presence of endothelial cell supernatants, and 14.6 ⫾ 0.3
nmoles in the presence of endothelial supernatant plus 1
unit/ml of adenosine deaminase. Superoxide release
from 105 nonstimulated neutrophils in the presence of
endothelial supernatants was 1.4 ⫾ 0.2 nmoles. This
indicated that adenosine was already present in supernatants derived from endothelial cells and that neutrophils were not supplying nucleotides (ATP or 5⬘-AMP)
for conversion to adenosine by endothelial-expressed
Figure 6. A, Effects of various concentrations of diisopropylfluorophosphate (DFP) on the release of von Willebrand factor (vWF) from endothelial
cells cultured with neutrophils that had been stimulated with fMLP (1 ␮M). Each well contained 105 neutrophils, and vWF was measured after 180
minutes of coculture. Analysis of variance (ANOVA) showed a significant effect of DFP treatment (P ⬍ 0.01). B, Effects of DFP on vWF release
by endothelial cells incubated with neutrophils and treated with antineutrophil cytoplasmic antibody (ANCA) IgG or fMLP. Neutrophils (105) were
untreated or were treated with normal IgG (200 ␮g/ml), ANCA IgG (200 ␮g/ml), or fMLP (1 ␮M) and were added to wells with unstimulated
endothelial cells in the presence or absence of DFP (5 mM). ANOVA showed a significant effect of treatments (P ⬍ 0.01 in each case). ⴱⴱ ⫽ P ⬍
0.01 versus untreated with DFP, by paired t-test. C, Dose response for effect of proteinase 3 (PR3) on the release of vWF by endothelial cells.
Release of vWF was measured after 180 minutes of treatment. ANOVA showed a significant effect of treatment on vWF release (P ⬍ 0.01). Values
are the mean and SEM of 5 experiments in A, 4 experiments in B, and 3 experiments in C.
Release of vWF from endothelial cells on exposure to neutrophils stimulated with fMLP or ANCA IgG.
Given that superoxide production from activated neutrophils was markedly inhibited by adenosine produced
by endothelial cells, we questioned whether the endothelial cells might still be injured by the neutrophils. The
release of vWF has been used as a marker of endothelial
cell injury, and circulating levels of vWF have been
shown to be increased in patients with acute vasculitis
(18–20). Hence, supernatants from neutrophil–
endothelial cell cocultures were assayed for vWF following stimulation of neutrophils with fMLP or ANCA IgG.
Both stimulants led to an increased release of vWF as
compared with vWF levels detected following exposure
of neutrophils and endothelial cells to normal IgG or to
no stimulant (Figure 4).
Inhibition of NADPH oxidase reduced production of neutrophil superoxide but did not inhibit release
of vWF from endothelial cells in cocultures. To examine
the role of any remaining superoxide production in the
release of vWF, we added the NADPH oxidase inhibitor
DPI to cocultures. In initial experiments, DPI (5 mM)
effectively reduced superoxide production from neutrophils alone that had been stimulated with fMLP. Superoxide production was 15.7 ⫾ 3.2 nmoles/105 neutrophils
(mean ⫾ SEM) in the absence of DPI and 1.3 ⫾ 0.3
nmoles/105 neutrophils in the presence of DPI.
In cocultures with endothelial cells, DPI also
reduced superoxide production. The mean ⫾ SEM
superoxide production was 5.8 ⫾ 0.9 nmoles/105 neutrophils without DPI (P ⬍ 0.05 compared with neutrophils
alone) and 0.95 ⫾ 0.25 nmoles/105 neutrophils with DPI
(P ⬍ 0.05 compared with neutrophils alone). However,
in coculture experiments with fMLP-stimulated neutrophils and endothelial cells, the release of vWF from
endothelial cells was not affected by DPI. The mean ⫾
SEM vWF release was 1.23 ⫾ 0.09 IU/dl in the absence
of DPI and 1.15 ⫾ 0.19 IU/dl in the presence of DPI.
Thus, neutrophil superoxide and other reactive
oxygen radicals whose production is dependent on
NADPH oxidase do not mediate the release of vWF
from endothelial cells. This observation was supported
by the lack of an inhibitory effect of combined treatment
with SOD (0.5 units/ml) and catalase (300 units/ml) on
vWF release from endothelial cells cocultured with
fMLP-stimulated or with ANCA IgG–stimulated neutrophils (data not shown).
Inhibited release of vWF from endothelial cells in
neutrophil–endothelial cell cocultures by inhibition of
serine proteases. In light of the above findings, we
considered whether proteolytic enzymes, rather than
oxidants, might be responsible for the release of vWF.
Stored supernatants from the same coculture experiments used for superoxide assay were analyzed. We
found that cocultured neutrophils released serine proteases after stimulation with fMLP and ANCA IgG, the
assay being sensitive to both elastase and PR3
(Figure 5).
We therefore added the serine protease inhibitor
DFP to neutrophil–endothelial cell cocultures. This led
to a dramatic dose-dependent reduction in the release of
vWF from endothelial cells cultured in the presence of
fMLP-stimulated neutrophils (Figure 6A). In further
experiments using DFP at a concentration of 5 mM,
DFP was shown to inhibit the vWF release in cocultures
of endothelial cells and neutrophils stimulated with
ANCA IgG or fMLP (Figure 6B). These observations
suggested that neutrophil serine proteases mediate the
release of vWF from endothelial cells cocultured with
activated neutrophils. Other investigators have previously shown that elastase can cause the release of vWF
from endothelial cells (21). We demonstrated here that
treatment of endothelial cells with PR3 induced the
release of vWF (Figure 6C).
Finally, we examined whether adenosine, which
could inhibit superoxide release, also affected protease
release. In neutrophils stimulated with fMLP or ANCA
IgG, the addition of adenosine (500 ␮M) had no significant effect on activity in supernatants. Protease activity
(absorbance) with ANCA IgG was 0.46 ⫾ 0.06 without
adenosine and 0.44 ⫾ 0.04 with adenosine (mean ⫾
SEM of 4 experiments). Protease activity with fMLP was
0.55 ⫾ 0.01 without adenosine and 0.50 ⫾ 0.01 with
adenosine (mean ⫾ SEM of 4 experiments).
This study explores the mechanisms by which
neutrophils may damage endothelial cells during adhesion and migration at sites of vasculitic lesions. Many in
vitro studies have demonstrated the ability of ANCA
IgG to stimulate cytokine-primed neutrophils to release
superoxide and serine proteases (PR3 and elastase) (for
review, see ref. 22). Both reactive oxygen radicals and
serine proteases have been proposed as initiators of
endothelial cell damage and vasculitic lesions in vivo.
Two major findings emerged from our study. First, the
presence of endothelial cells inhibits the release of
superoxide by neutrophils via mechanisms that are dependent on adenosine. Second, serine proteases, rather
than superoxide, appeared to be the important effector
of endothelial vWF release, which was used as a marker
of endothelial cell injury. This was true irrespective of
whether neutrophils were activated by fMLP or by
Adenosine has been recognized for many years to
be an inhibitor of superoxide and hydrogen peroxide
release from neutrophils following stimulation with
fMLP but in the absence of endothelial cells (23), and to
be an inhibitor of neutrophil-mediated injury to endothelial cells (17). Other investigators have shown that the
adhesion of neutrophils to surfaces coated with various
extracellular matrix proteins (fibronectin, fibrinogen,
laminin) caused a delayed respiratory burst in response
to soluble stimuli, such as fMLP and TNF␣ (24); in those
studies, a respiratory burst still developed after a delay
of 30–90 minutes.
In our studies, we found that neutrophils rolling
on P-selectin were able to mount a respiratory burst
after stimulation with fMLP and ANCA IgG, which
concurrently induced stationary adhesion. In this regard,
P-selectin model is a useful surrogate for an endothelial
surface, and adhesion in the flow system effectively
primes the neutrophils; we anticipated that superoxide
release would occur for activated neutrophils on endothelium in the flow system through similar adhesive
priming. However, although fMLP and ANCA IgG
induced superoxide release when neutrophils were adherent to P-selectin, these substances failed to induce a
detectable respiratory burst when neutrophils were rolling on activated endothelial cells, even though the assay
was essentially the same and the neutrophils developed
stationary adhesion. The differences in the respiratory
burst could not be attributed to differences in neutrophil
numbers. The quantities of superoxide released from
neutrophils rolling over P-selectin were indeed lower
than in the prolonged static incubations, where endothelial contact greatly inhibited release, but superoxide was
still detectable. Thus, we conclude that superoxide release from the activated neutrophils adherent to endothelium in the flow system was greatly reduced and that
any residual release then fell below the detectable level.
We cannot conclude that there was zero release.
Using a static coculture model, the effect of
endothelial cell interactions on the neutrophil superoxide response to the chemoattractant fMLP and to
ANCA IgG was examined. Endothelial cells inhibited
superoxide production, and this was greatest when the
endothelial cells had not been activated with the cytokine TNF␣; unlike the effects with extracellular matrix
proteins, the inhibitory response was maintained over
180 minutes. Adenosine was then investigated as a
possible mediator of these effects. We confirmed the
earlier observations that adenosine inhibits stimulated
neutrophil superoxide release (23); use of the adenosine
receptor antagonist 8-SPT confirmed that this was likely
to be operating via adenosine receptors. Addition of
adenosine deaminase to neutrophil–endothelial cell cocultures restored superoxide responses to baseline levels
(i.e., levels produced by stimulated neutrophils alone),
confirming that adenosine was mediating the inhibition.
Intracellular or extracellular adenosine 3⬘,5⬘-monophosphate may be converted to adenosine through dephosphorylation by cytosolic- and ecto-5⬘-nucleotidases
(25), with CD73, the interferon-␣–inducible endothelial
ecto-5⬘-nucleotidase, being a potent converter of AMP
to adenosine (26). Although we noted a lesser effect of
TNF␣-activated endothelium on the suppression of neutrophil superoxide release, TNF␣ has no apparent effects on the level of CD73 expression on endothelial
cells (26). Adenosine may be important for reducing,
albeit not preventing, endothelial damage in the event
that activated neutrophils remain in close contact with
endothelium for prolonged periods.
The observed down-regulation of superoxide production suggested that this neutrophil product might not
be an inducer of endothelial cell injury. We addressed
this using vWF release as a surrogate marker of endothelial cell injury. Serum levels of vWF are often used as
a marker of disease activity and endothelial cell injury in
the ANCA-associated vasculitic diseases (18–20). That
reactive oxygen species were not mediators of endothelial injury was supported by the inability of DPI (and of
the combination of SOD and catalase) to inhibit vWF
release in coculture experiments with fMLP- or ANCA
IgG–stimulated neutrophils. Since fMLP and ANCA
IgG can induce neutrophil degranulation with release of
the serine proteases PR3 and elastase, these were tested
as potential mediators of the neutrophil-induced vWF
release using the potent serine protease inhibitor DFP.
The ability of DFP to prevent the fMLP- and ANCA
IgG–associated release of vWF in coculture studies
strongly supports the serine proteases as mediators of
vWF release from endothelial cells. This was further
substantiated by the ability of PR3 in our studies and
elastase in previous studies (21) to induce vWF release
when added directly to endothelial cells, as well as by our
detection of protease activity in supernatants of
neutrophil–endothelial cell cocultures treated with
Elastase and PR3 have been shown to be potent
inducers of endothelial cell apoptosis in vitro. These
serine proteases can be taken up into endothelial cells
and can cleave p65 NF-␬B (27). In the case of PR3,
there is sustained JNK activation and cleavage of
p21 WAF1/Cip1/Sdi1 (28). In patients with ANCAassociated vasculitis, circulating levels of PR3 are increased, which adds to the likelihood that serine proteases released from neutrophils are key initiators of
endothelial injury in these disorders. Nonetheless, it
would be prudent to exercise some caution in extrapolating to the in vivo situation the findings from our
model system, given that it was blood-free and used
macrovascular, rather than microvascular, endothelial
cells. ANCA-associated vasculitis predominantly affects
capillary endothelial cells, and HUVECs may not be
fully representative of pulmonary or glomerular capillary endothelial cells.
In summary, we have confirmed previous reports
that neutrophil superoxide release can be inhibited by
adenosine, and we have shown that inhibition of neutrophil superoxide production occurs during neutrophil–
endothelial cell coculture through this mediator. In
addition, we have shown the importance of these effects
by demonstrating that they are operative when neutrophils are stimulated by pathologic agonists, such as
ANCA IgG. Our study is the first to show that serine
proteases released from ANCA IgG– or fMLPstimulated neutrophils can induce endothelial vWF release. We suggest that this adds to the growing evidence
that serine proteases are important initiators of endothelial injury in the ANCA-associated vasculitides.
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serine, endothelial, cytoplasmic, neutrophils, antineutrophil, release, superoxide, mediation, damage, protease, antibodystimulated, cells
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