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Cardiovascular Biology Research, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104; and
Howard Hughes Medical Institute, Oklahoma City, OK 73104
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß cytokines (14, 15). Thus, PR3 expression near a vascular surface would very
likely contribute to local tissue destruction and inflammation. In ongoing studies with the primate model of Escherichia coli-mediated septic shock, we found that blocking the endothelial protein C receptor (EPCR) in vivo with specific mAbs transformed a transient response to sublethal E. coli into a lethal response (16). EPCR is a type 1 transmembrane protein expressed on endothelium that binds protein C zymogen and facilitates formation of activated protein C, a potent anticoagulant (17) and antiinflammatory protein (18, 19). A soluble form of EPCR, which exists in plasma, retains full ligand-binding capability (20), and levels increase in patients with sepsis or systemic lupus erythematosus (21) either from vascular injury or through a regulated proteolytic release of soluble receptor (22). Of particular interest in the primate study was the observation of a massive polymorphonuclear cell influx into specific areas of the adrenal, liver, and kidney, suggesting that EPCR may be involved in neutrophil interactions and leukocyte trafficking.
In the present study, we examined whether EPCR could interact directly with neutrophils. It was found that recombinant soluble EPCR (sEPCR) binds to activated neutrophils via PR3 and supported, in part, by CD11b/CD18 (Mac-1), a ß2 integrin involved in cell-cell adhesion and neutrophil-signaling events. The results are discussed with respect to their potential importance in modulating vascular damage due to inflammatory stimuli.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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sEPCR (3) was a recombinant soluble human EPCR and was prepared as described previously (23). The construct codes for the extracellular domain of EPCR truncated immediately above the transmembrane domain at residue 210, with a 12-residue HPC4 epitope tag at the carboxyl terminus for calcium-dependent HPC4-immunoaffinity purification (24). sEPCR was labeled with Oregon Green 488 carboxylic acid, succinimidyl ester 5-isomer (Molecular Probes, Eugene, OR), or Cy3 (Pharmacia-Amersham, Uppsala, Sweden) using standard methods. Human neutrophil PR3 was obtained from Athens Research and Technology (Athens, GA) and biotinylated with sulfo-NHS-LC-biotin (sulfosuccinimidyl 6-(biotinamido)hexanoate; Pierce, Rockford, IL).
Monoclonal Abs
The preparation of and screening methods for mAbs against human
EPCR have been described elsewhere (20). The PL1 mAb
against neutrophil P-selectin glycoprotein ligand-1 was a kind gift
from Kevin Moore (Cardiovascular Biology Research, Oklahoma Medical
Research Foundation, Oklahoma City, OK). The IB4 (IgG2a) hybridoma cell
line was obtained from American Type Culture Collection (Manassas, VA),
and the TS1 (IgG1) hybridoma cell line was obtained from the
Developmental Studies Hybridoma Bank, Department of Biological
Sciences, University of Iowa (Iowa City, IA). Abs were purified from
hybridoma supernatants by protein G affinity chromatography using
standard methods. mAbs against CD11a (clone HI111; IgG1) and CD11b
[clone ICRF44 (44); IgG1] were purchased from PharMingen (San Diego,
CA). Hybridoma supernatant containing Ab against CD11c (IgG1) was
obtained from Serotec (Oxford, U.K.). The Ab preparations against CD11b
and CD11c -chains were reported by their respective manufacturers to
inhibit ß2 integrin-mediated adhesion
functions. V189 (IgG2a), an anti-human factor V mAb, served as an
isotype control for IB4.
Blood collection and neutrophil preparation
Blood was collected by venipuncture into vacutainer tubes (Becton Dickinson, San Jose, CA) containing heparin (100 U.S.P. units), buffered sodium citrate (3.8%), or EDTA (7.5% K3EDTA). For anticoagulation with hirudin (Sigma, St. Louis, MO), blood was collected into a sterile tube containing 50 U/ml hirudin and mixed.
For experiments using purified neutrophils, heparinized blood was mixed
with an equal volume of 6% Dextran 70 in 0.9% NaCl (McGaw, Irvine,
CA). RBC were allowed to sediment for 45–60 min at room temperature.
The supernatant was removed and centrifuged at 400 x g
(10 min, 4°C). The pellet was resuspended in 25 ml of ice-cold 0.2%
NaCl with mixing for 25 s, followed by 25 ml of ice-cold 1.6%
NaCl. The cells were centrifuged and the pellet gently resuspended in 5
ml of HHB buffer (HBSS, 10 mM HEPES, pH 7.5, 1% BSA). This was
transferred to a 15-ml conical centrifuge tube and underlayered with 5
ml of Lymphocyte Separation Media (density 1.077 ± 0.001 g/ml at
22°C; Cellgro, Herndon, VA). The sample was centrifuged at 400
x g for 30 min at 4°C. The pellet containing purified
neutrophils was resuspended in 5–10 ml HHB buffer. A typical yield was
10 x 106 neutrophils per
preparation.
sEPCR affinity chromatography
Leukocytes were purified from 50 ml of citrated blood by
sedimentation through dextrose and hypotonic lysis, as described above.
The cell pellet was washed and resuspended in HBSS, 10 mM HEPES (pH
7.5), 3 mM CaCl2, and 0.6 mM
MgCl2 and surface biotinylated with 0.5 mg/ml
sulfo-NHS-LC-biotin for 30 min at room temperature. The cells were
washed, then lysed with 1% Nonidet P-40 in 10 mM HEPES (pH 7.5) and
0.1 mM EDTA, and centrifuged. The supernatant was mixed with 100 µl
of sEPCR-AffiGel 10 (Bio-Rad; 1 mg sEPCR/ml resin) or
Tris-inactivated AffiGel 10 (control) in HHB/CaMg buffer (HHB with 3 mM
CaCl2, 0.6 mM MgCl2; total
volume 600 µl). The samples were mixed overnight at 4°C, washed
five times with buffer (without albumin), and eluted with HBSS, 50 mM
HEPES (pH 7.5) and 5 mM EDTA. The samples were centrifuged and the
supernatant was analyzed by SDS-PAGE on a 4–20% gradient gel. The
samples were transferred to a polyvinylidene difluoride membrane, and
the membrane was blocked with a 10% (w/v) nonfat dry milk solution,
washed, and incubated with streptavidin-alkaline phosphatase conjugate.
Bound conjugate was detected with ECF substrate (Amersham-Pharmacia),
and image analysis was performed using a Storm 860 imager (Molecular
Dynamics, Sunnyvale, CA).
The procedure was scaled up so that purified neutrophils from 400 ml
citrated blood were washed and lysed with 1% Nonidet P-40, 10 mM HEPES
(pH 7.5), and 0.1 mM EDTA. The lysate was centrifuged, and the
supernatant was diluted 5-fold and adjusted to 3 mM CaCl2,
0.6 mM MgCl2. This was applied to an
sEPCR-Affi-Gel 10 affinity column (25 ml of resin coupled at 1 mg
sEPCR/ml resin) equilibrated in 20 mM Tris-HCl, 0.1 M NaCl, 0.2%
Nonidet P-40, 3 mM CaCl2, and 0.6 mM
MgCl2 (pH 7.5), with a 10-ml precolumn of
Tris-inactivated Affi-Gel 10 to reduce nonspecific interactions. The
cell pellet was extracted twice more with lysing buffer, and the
supernatants were applied. The column was washed to baseline
OD280 and eluted with buffer containing 50 mM
HEPES (pH 7.5) and 5 mM EDTA. The eluate fractions were pooled,
concentrated, and analyzed by SDS-PAGE on a 10% resolving gel. The
band corresponding to the 33-kDa band observed previously was cut out
and sent to the Harvard Microchemistry Facility (Cambridge, MA) for
matrix-assisted laser desorption/ionization mass spectrometry
(MALDI-MS) analysis, trypsin digestion, and amino-terminal
sequencing.
Flow cytometry
Data acquisition by flow cytometry and subsequent analysis was done with a FACScaliber flow cytometer using CellQuest software (Becton Dickinson, San Jose, CA). Neutrophils were gated according to their relative size (forward scatter) and relative granularity (side scatter) properties.
sEPCR binding to activated neutrophils
For experiments using blood, 50-µl aliquots were incubated at 37°C for 10 min with 500 nM Oregon Green-sEPCR and 0.1 µg/ml PMA (Sigma). Samples were placed on ice, and RBC were lysed with 1 ml FACS Lysing Solution (Becton Dickinson). After centrifugation, pellets were resuspended in 1 ml of ice-cold HHB/CaMg buffer and centrifuged. The washed pellet was resuspended in ice-cold HHB/CaMg buffer and analyzed by flow cytometry. Purified neutrophils were evaluated for sEPCR binding in a similar fashion, with the exception of the lysing solution.
sEPCR binding to neutrophils in the presence of Abs against ß2 integrins
Heparinized blood (50 µl) was preincubated at room temperature for 15 min with purified Ab (IB4, TS1, PL1, V189, anti-CD11a, anti-CD11b, anti-CD11c), 10 µl of anti-CD11c hybridoma culture supernatant, or 10 µl of nonconditioned media. The final concentration of the purified Abs was 40 µg/ml. Oregon Green-sEPCR (500 nM) was added in the absence or presence of PMA (100 nM), and the samples were incubated at 37°C for 15 min. RBC were removed by hypotonic lysis, the cells were washed, and cell-associated fluorescence was determined by flow cytometry, as described above.
Microscopy
The PR3-ANCA, perinuclear ANCA (P-ANCA), and sEPCR interaction with ethanol-fixed neutrophils was evaluated with a Leica TCS NT confocal system equipped with four-laser excitation, four fluorescent detectors, and a transmitted detector (Heidelberg, Germany). These studies were performed at the Flow and Image Cytometry Laboratory, University of Oklahoma Health Sciences Center, William K. Warren Medical Research Institute (Oklahoma City, OK). Slides with ethanol-fixed neutrophils, P-ANCA, and FITC anti-human IgG were from The Binding Site (Birmingham, U.K.). PR3-ANCA was from the Varelisa Autoimmune Analysis kit (Pharmacia & UpJohn, Kalamazoo, MI). The cells were incubated with the autoantibodies (30 min) and washed with HHB buffer. Bound Ab was detected with FITC anti-human IgG reagent according to the manufacturer’s directions. The slides were washed once in HHB and twice in HHB/CaMg, and then incubated with Cy3-sEPCR (1 µM) in HHB/CaMg buffer (30 min). The slides were washed with HHB/CaMg and mounted with a coverslip and Slow Fade reagent (Molecular Probes). The slides were then visualized for FITC and Cy3 fluorescence.
Binding of PR3 and sEPCR
Microtiter plate wells were coated overnight at 4°C with 4 µg/ml of mAbs against sEPCR (1494, 1500, or HPC4 at 50 µl/well). All subsequent steps were done at room temperature. The wells were washed with wash buffer (20 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 0.05% Tween 20, 3 mM CaCl2, 0.6 mM MgCl2), blocked, washed again, and then incubated with 100 nM sEPCR (50 µl/well) for 30 min. This approach was used to create a binding surface because EPCR tends to be conformationally sensitive and, at least for screening mAbs, immobilization of EPCR directly to plastic surfaces destroys a majority of the available epitopes. After washing, increasing concentrations of biotin-PR3 were added (30 min). The plates were washed, and bound biotin-PR3 was detected with streptavidin-alkaline phosphatase (1 µg/ml, 30 min) and Blue Phos substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Color development was stopped with 2.5% EDTA, and the absorbance at 650 nm was read in a Vmax microplate reader (Molecular Devices, Sunnyvale, CA). The background absorbance observed in the absence of coating Ab was subtracted from all samples.
Microtiter plates precoated with human PR3 (The Binding Site) were used to assess the ability of excess unlabeled sEPCR to inhibit binding of biotin-sEPCR to purified PR3. sEPCR (0–5 µM) was added to the PR3-coated wells (30 min, room temperature), followed by 500 nM biotin-sEPCR (30 min). The wells were washed (HHB/CaMg), and bound biotin-sEPCR was detected with streptavidin-alkaline phosphatase and Blue Phos substrate, as described above.
Normal sera and patient sera containing anti-PR3 Abs
Sera from five patients were kindly provided for this study by Judith James-Wood (Arthritis & Immunology Department, Oklahoma Medical Research Foundation). Four of the patients (P1, P2, P3, and P5) have biopsy-proven Wegener’s granulomatosis, as well as anti-PR3 autoantibodies by commercial ELISA (PR3-ANCA), and were not on aggressive immunosuppressive therapy at the time of collection. The fifth patient (P4) has anti-PR3 autoantibodies, but only eye involvement and not systemic disease, and does not fulfill the criteria for Wegener’s. These samples were used to evaluate the influence of serum containing anti-PR3 autoantibodies on the binding of sEPCR and PR3 in vitro. Normal sera were obtained from three apparently healthy adult volunteers (N1, N2, and N3) using standard venipuncture techniques. IgG was purified for some experiments on protein G columns using standard methods.
Influence of sera on sEPCR binding to PR3
Serum samples were diluted 1/200 and 1/1000 and preincubated for 30 min at room temperature on plates precoated with PR3 (The Binding Site). Biotinylated-sEPCR was added (80 nm final), and incubation continued for an additional 30 min. The plates were washed and bound biotin signal detected with streptavidin-HRP and tetramethylbenzidine substrate. The absorbance at 450 nm was read on a Vmax microplate reader. The maximal binding (100%) was the level of bound sEPCR after preincubation of the PR3 wells with buffer (10 mM HEPES, pH 7.4, 0.1 M NaCl, 0.05% Tween 20) instead of serum.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the fluorescently labeled sEPCR bound
to PMA-activated neutrophils. Without activation (no PMA), the
cell-associated fluorescence was only slightly higher than the
background cell autofluorescence (neutrophils alone). In contrast, the
fluorescence associated with the PMA-activated neutrophils (solid
black) was at least an order of magnitude higher than the controls.
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). Essentially the same results
were obtained, and activation of the neutrophils again greatly enhanced
sEPCR binding. An EDTA buffer system supported about half the sEPCR
binding relative to the others, the difference with citrate probably
attributable to the stronger chelating properties of EDTA. These
experiments were repeated multiple times with blood from three
different donors with essentially identical results.
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, lane 2). More bands were observed with longer exposure
times, but correlated with those in the control sample
(lane 1).
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30 kDa), a serine proteinase stored in the
secretory vesicles and primary and azurophilic granules of neutrophils.
This sequence of PR3 is distinct from the homologous region of
neutrophil elastase, azurocidin, or cathepsin G, which are closely
related structurally and also stored in azurophilic granules
(25).
To test directly whether sEPCR was binding to PR3, in vitro studies
were done using purified components. To create a binding surface,
microtiter plate wells were coated with mAbs to EPCR, and the Ab
surface was then saturated with sEPCR. This approach was used to create
a binding surface because direct immobilization of EPCR on plastic
surfaces was found to destroy the conformation of EPCR. Purified
biotin-PR3 was then added and, in each case, the Ab-sEPCR surface
supported biotin-PR3 binding in a saturable manner (Fig. 4A). These are nonoverlapping
Abs that either block protein C/activated protein C binding to EPCR
(1494) (17, 26), do not block ligand binding (1500), or
bind to the carboxyl-terminal tag (HPC4). None of the anti-sEPCR
mAbs screened to date appear to inhibit the sEPCR-PR3 interaction. In
addition, increasing concentrations of unlabeled sEPCR decreased
biotin-sEPCR binding to PR3-coated wells (Fig. 4B). Thus,
the sEPCR-PR3-binding interaction was saturable and dissociable with
excess, unlabeled ligand using purified components.
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This technique was used in two-color confocal microscopy colocalization
studies with ethanol-fixed neutrophils (Fig. 5). sEPCR was labeled with Cy3, a
fluorescent probe that emits a red color. The PR3-ANCA (or P-ANCA) was
detected with FITC anti-human IgG that emits a green color. When
the two signals overlap, a yellow color is observed. Cy3-sEPCR staining
was diffuse and distributed throughout the cytoplasm (Fig. 5, A and E), and the PR3-ANCA produced the typical
green cytoplasmic staining (Fig. 5B). When the images were
overlaid, extensive areas of diffuse, yellow color were observed in the
cell cytoplasms (Fig. 5C). In contrast, similar experiments
with P-ANCA (Fig. 5D) and Cy3-sEPCR (Fig. 5E) did
not show colocalization (Fig. 5F). Thus, the sEPCR localized
to neutrophil cytoplasmic sites in parallel with the PR3-ANCA, whose
target Ag is PR3.
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). The PR3 was immobilized
on the surface of microtiter plate wells and preincubated with buffer
(100%), normal serum samples (N1, N2, and N3), or patient sera
containing PR3-ANCA (P1-P5). Although the normal serum samples did not
affect sEPCR binding to PR3, preincubation with three of the patient
sera diluted 1/200 resulted in reduced sEPCR binding (P2, P3, and P4).
Further dilution of these sera to 1/1000 resulted in sEPCR binding
similar to the normal samples. Incubation with the P1 and P5 sera did
not appreciably alter sEPCR binding to the PR3-coated wells. The
relative ability to reduce sEPCR binding did not correlate with the
PR3-ANCA titer of the patient samples (data not shown) and is more
likely related to the PR3 epitope specificity of the autoantibodies. A
similar result was observed using confocal microscopy in which
pretreatment of the ethanol-fixed neutrophils with PR3-ANCA, but not
P-ANCA (positive and negative control sera with the commercial PR3
ELISA), reduced the signal observed from Oregon Green-labeled sEPCR
binding by 50%, relative to untreated cells (data not shown).
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1-antitrypsin (a natural inhibitor of PR3 protease activity)
significantly alters the sEPCR-PR3 interaction (data not shown).
The ability of serum containing PR3-ANCA to reduce sEPCR binding to PR3
raised the possibility that sEPCR may modulate the protein-protein
interactions in this system. Although it is generally accepted that
PR3-ANCA binds to neutrophil PR3, it is less widely appreciated that
accessory molecules may be involved as well. A previous study indicated
that elastase and azurocidin, and possibly PR3, are ligands for
the ß2 integrin, Mac-1 (CD11a/CD18), on the
neutrophil surface (29). Mac-1 is a member of the
ß2 integrin family, consisting of LFA-1
(CD11a/CD18), Mac-1 (CD11b/CD18), p150,95 (CD11c/CD18), and a newly
described fourth member (d/CD18)
(30). These are heterodimeric complexes with a common
ß-chain (CD18) and unique -chains (CD11a, b, c, and
d), and are involved in neutrophil adhesion
and signaling (30, 31, 32).
These observations raised the possibility that sEPCR may be binding to
a preassembled PR3-ß2 integrin complex on the
cell surfaces. To address this, sEPCR binding to neutrophils activated
in whole blood was evaluated in the presence of buffer or mAbs to the
ß2 integrins (Fig. 7). IB4 Ab (IgG2a) binds to the common
ß-chain of the ß2 integrin family (CD18) and
blocks ß2 integrin-mediated adhesion
(33). This Ab reduced sEPCR binding to PMA-activated
neutrophils by 81% (p < 0.0001).
Interestingly, TS-1 Ab (IgG1) had either no effect, or slightly
increased, sEPCR binding to the cells. TS-1 also binds to CD18, but to
a different epitope, and does not inhibit neutrophil adhesion to matrix
proteins. As controls, PL-1 (IgG1), which binds to neutrophil
P-selectin glycoprotein ligand-1 (34), had no effect on
sEPCR binding, nor did an irrelevant isotype-matched Ab (V189; IgG2a).
Essentially identical results were observed when IB4 was added to
purified neutrophils and sEPCR binding to PMA-activated neutrophils was
determined (data not shown).
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). This
indicates that at least Mac-1 is involved in the neutrophil receptor
complex that supports sEPCR binding, probably as a PR3/Mac-1
heterocomplex. The question then arose as to whether IB4, the anti-CD18-blocking Ab, could alter PR3-ANCA IgG binding to activated neutrophils. Neutrophils were preincubated with IB4 (10 µg/ml) in the absence or presence of each patient PR3-ANCA IgG (100 µg/ml) and activated with cytochalasin B/fMLP, and bound Ab was detected with fluorescein-anti-human IgG by flow cytometry. There was no difference in the amount of patient IgG bound in the presence of IB4 Ab (data not shown). The simplest interpretation is that the ability of IB4 to block sEPCR binding to the cells (and presumably to PR3) is a steric effect, rather than a direct inhibition of a ligand interaction. Interestingly, IgG from P1 and P5, which did not reduce sEPCR binding to activated neutrophils, also bound very little to the activated neutrophils relative to the other three autoantibodies.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although a causative role for the PR3-ANCA autoantibodies in the
etiology of autoimmune vasculitis remains controversial, many in vitro
studies have demonstrated that they are capable of propagating an
inflammatory response by cross-linking Fc
receptors as well as by
PR3 binding to F(ab')2 regions
(35, 36, 37, 38, 39, 40). In Wegener’s patients, PR3 is expressed on the
surface of circulating neutrophils (41, 42), and PR3-ANCA
binding to PR3 on the surface of TNF--primed neutrophils sets off a
full-blown activation response, including degranulation and production
of oxygen radicals (35). However, mechanisms that may
modulate this process of vasculitis are poorly understood. The current
study suggests that sEPCR may modulate the ability of PR3-ANCA to bind
to PR3 on neutrophils, potentially regulating the inflammatory response
and vasculitic injury. However, this potential regulatory role is
speculative and it must be recognized that there is no in vivo data
defining a pathogenic role for ANCA, and the possibility remains that
the presence of ANCA in the vasculitis patients is an
epiphenomenona.
One prerequisite for the current model of vascular injury is local
accumulation of primed neutrophils that will eventually adhere to the
endothelium to provide a local source of inflammatory mediators. The
ß2 integrins, which include Mac-1 (CD11b/CD18),
are expressed on granulocytes and monocytes and are involved in
cell-cell adhesion and signaling events. Integrin expression is
inducible, and circulating leukocytes from patients with active
Wegener’s granulomatosis show significantly increased surface
expression of Mac-1 that declines to normal levels upon treatment
(43). Expression of two other ß2
integrins (LFA-1 and p150,95) was normal in the Wegener’s patients, as
was Mac-1 expression on cells from patients with other systemic
diseases (systemic lupus erythematosus, myeloperoxidase-positive
systemic vasculitis, sepsis). An additional study found that CD18 on
TNF--primed neutrophils was required for the respiratory burst
activation induced by an anti-PR3 mAb (40). These
observations are consistent with the current data demonstrating Mac-1
participation in the sEPCR binding site on neutrophils, possibly as a
PR3/Mac-1 heterocomplex (29). A recent study further
demonstrated that PR3-ANCA was capable of a transient activation of
rolling neutrophils under flow conditions with resultant firm adhesion
to a platelet selectin surface (44). This activation and
adhesion was Mac-1 mediated and dependent on interaction of the ANCA
with Fc receptors. One prediction from our working model is that
sEPCR may modulate Mac-1 integrin-mediated events, such as outside-in
signaling or adhesion, and these studies are in progress. Based on
current models, neutrophils play a role in the early stages of
vasculitic injury, and the later fulminant lesions typically consist of
lymphocytes and macrophages.
The current studies demonstrating Mac-1 participation in the binding complex for sEPCR are uniquely limited by the epitope specificity of the Abs used. Mac-1 has a diverse ligand repertoire (ICAM-1, fibrinogen, complement iC3b, coagulation factor X, neutrophil elastase, azurocidin, PR3), and there is no particular reason to believe that PR3 has a singular preference for binding to only one ß2 integrin. It is quite possible that the epitopes of the Abs against CD11a and CD11c may not have overlapped the ligand binding site. Additional studies with other Abs are ongoing to further evaluate LFA-1 or p150,95 as potential partners in the sEPCR-binding complex. Participation of Mac-1 in the sEPCR-binding complex does provide insight into the partial metal dependence observed for sEPCR binding to the activated neutrophils since ß2 integrin function is metal ion dependent (45).
The current studies were done with sEPCR, raising the question of whether EPCR anchored in the endothelial membrane shares this ability to bind PR3. In this regard, EPCR mRNA levels increase rapidly in response to endotoxin challenge in a rat model of septic shock (22). Although the tissue EPCR Ag levels do not rise appreciably, there is a substantial increase in sEPCR levels in the plasma, suggesting a regulated pathway for EPCR synthesis and sEPCR release, possibly through thrombin stimulation of the endothelium and subsequent metalloproteinase activity (46). This concept is supported by observations that significantly elevated levels of circulating soluble EPCR are observed in patients with sepsis or systemic lupus erythematosus (21). Regulated release of soluble EPCR may play a role in Wegener’s patients in whom selective microvascular beds in the nose, sinuses, lungs, and kidneys are the initial targets of the vasculitis (7), sites in which membrane-bound EPCR expression is relatively poor (47). However, unlike its membrane-bound parent, soluble EPCR is not restricted to a particular vascular bed and could be recruited for binding to neutrophils.
These observations represent novel additions toward understanding the fundamental mechanisms of inflammation and the molecular basis for the vasculitis in Wegener’s granulomatosis and related vasculitides. Additional studies are required to further characterize the effect of PR3-ANCA and other modulators on the pair-wise interactions between PR3, soluble EPCR, and CD11b/CD18 on activated neutrophils.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Shinichiro Kurosawa, Free Radical Biology and Aging Department, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104.
3 Current address: Free Radical Biology & Aging Department, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104.
4 Abbreviations used in this paper: PR3, proteinase-3; ANCA, anti-neutrophil cytoplasmic Ab; EPCR, endothelial protein C receptor; P-ANCA, perinuclear ANCA; sEPCR, recombinant soluble EPCR.
Received for publication September 17, 1999. Accepted for publication July 24, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IL-1ß from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3. Proc. Natl. Acad. Sci. USA 96:6261.. J. Biol. Chem. 270:23688.M ß2) and modulates polymorphonuclear leukocyte adhesion. J. Exp. Med. 184:1213.-d ß-2, binds preferentially to ICAM-3. Immunity 683.
1-antitrypsin, and Fc receptors. J. Clin. Invest. 100:1416.[Medline]
receptor II-mediated signal transduction: relevance for activation of neutrophils by anti-proteinase 3 or anti-myeloperoxidase antibodies. Blood 86:3189.This article has been cited by other articles:
|
|
 |
|
  S. Gandrille Endothelial cell protein C receptor and the risk of venous thrombosis Haematologica, June 1, 2008; 93(6): 812 - 816. [Full Text] [PDF]
|
|
|
|
 |
|
 B. Saposnik, E. Lesteven, A. Lokajczyk, C. T. Esmon, M. Aiach, and S. Gandrille Alternative mRNA is favored by the A3 haplotype of the EPCR gene PROCR and generates a novel soluble form of EPCR in plasma Blood, April 1, 2008; 111(7): 3442 - 3451. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. O. Mosnier, B. V. Zlokovic, and J. H. Griffin The cytoprotective protein C pathway Blood, April 15, 2007; 109(8): 3161 - 3172. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Thiyagarajan, T. Cheng, and B. V. Zlokovic Endothelial Cell Protein C Receptor: Role Beyond Endothelium? Circ. Res., February 2, 2007; 100(2): 155 - 157. [Full Text] [PDF]
|
|
|
|
|
|
X. Zheng, W. Li, J.-M. Gu, D. Qu, G. L. Ferrell, N. L. Esmon, and C. T. Esmon Effects of membrane and soluble EPCR on the hemostatic balance and endotoxemia in mice Blood, February 1, 2007; 109(3): 1003 - 1009. [Abstract] [Full Text] [PDF]
|
|
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|
 |
|
 M. J. Ludeman, H. Kataoka, Y. Srinivasan, N. L. Esmon, C. T. Esmon, and S. R. Coughlin PAR1 Cleavage and Signaling in Response to Activated Protein C and Thrombin J. Biol. Chem., April 1, 2005; 280(13): 13122 - 13128. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Iwaki, D. T. Cruz, J. A. Martin, and F. J. Castellino A cardioprotective role for the endothelial protein C receptor in lipopolysaccharide-induced endotoxemia in the mouse Blood, March 15, 2005; 105(6): 2364 - 2371. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. V. de Wouwer, D. Collen, and E. M. Conway Thrombomodulin-Protein C-EPCR System: Integrated to Regulate Coagulation and Inflammation Arterioscler Thromb Vasc Biol, August 1, 2004; 24(8): 1374 - 1383. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Opal The nexus between systemic inflammation and disordered coagulation in sepsis Innate Immunity, April 1, 2004; 10(2): 125 - 129. [Abstract] [PDF]
|
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|
|
 |
|
 A. David, Y. Kacher, U. Specks, and I. Aviram Interaction of proteinase 3 with CD11b/CD18 ({beta}2integrin) on the cell membrane of human neutrophils J. Leukoc. Biol., October 1, 2003; 74(4): 551 - 557. [Abstract] [Full Text] [PDF]
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 C. T. Esmon The Protein C Pathway Chest, September 1, 2003; 124 (2009): 26S - 32S. [Abstract] [Full Text] [PDF]
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D. H. Sturn, N. C. Kaneider, C. Feistritzer, A. Djanani, K. Fukudome, and C. J. Wiedermann Expression and function of the endothelial protein C receptor in human neutrophils Blood, August 15, 2003; 102(4): 1499 - 1505. [Abstract] [Full Text] [PDF]
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C. T. Esmon Coagulation and inflammation Innate Immunity, June 1, 2003; 9(3): 192 - 198. [Abstract] [PDF]
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 G. A. Preston, C. S. Zarella, W. F. Pendergraft III, E. H. Rudolph, J. J. Yang, S. B. Sekura, J. C. Jennette, and R. J. Falk Novel Effects of Neutrophil-Derived Proteinase 3 and Elastase on the Vascular Endothelium Involve In Vivo Cleavage of NF-{kappa}B and Proapoptotic Changes in JNK, ERK, and p38 MAPK Signaling Pathways J. Am. Soc. Nephrol., December 1, 2002; 13(12): 2840 - 2849. [Abstract] [Full Text] [PDF]
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J.-M. Gu, J. T. B. Crawley, G. Ferrell, F. Zhang, W. Li, N. L. Esmon, and C. T. Esmon Disruption of the Endothelial Cell Protein C Receptor Gene in Mice Causes Placental Thrombosis and Early Embryonic Lethality J. Biol. Chem., November 1, 2002; 277(45): 43335 - 43343. [Abstract] [Full Text] [PDF]
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 C. T. Esmon New Mechanisms for Vascular Control of Inflammation Mediated by Natural Anticoagulant Proteins J. Exp. Med., September 2, 2002; 196(5): 561 - 564. [Full Text] [PDF]
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V. Oganesyan, N. Oganesyan, S. Terzyan, D. Qu, Z. Dauter, N. L. Esmon, and C. T. Esmon The Crystal Structure of the Endothelial Protein C Receptor and a Bound Phospholipid J. Biol. Chem., July 5, 2002; 277(28): 24851 - 24854. [Abstract] [Full Text] [PDF]
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A. Y. Kim, P. L. Walinsky, F. D. Kolodgie, C. Bian, J. L. Sperry, C. B. Deming, E. A. Peck, J. G. Shake, G. B. Ang, R. H. Sohn, et al. Early Loss of Thrombomodulin Expression Impairs Vein Graft Thromboresistance: Implications for Vein Graft Failure Circ. Res., February 8, 2002; 90(2): 205 - 212. [Abstract] [Full Text] [PDF]
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D. J. Stearns-Kurosawa, K. Swindle, A. D'Angelo, P. Della Valle, A. Fattorini, N. Caron, M. Grimaux, B. Woodhams, and S. Kurosawa Plasma levels of endothelial protein C receptor respond to anticoagulant treatment Blood, January 15, 2002; 99(2): 526 - 530. [Abstract] [Full Text] [PDF]
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 Z. G. Laszik, X. J. Zhou, G. L. Ferrell, F. G. Silva, and C. T. Esmon Down-Regulation of Endothelial Expression of Endothelial Cell Protein C Receptor and Thrombomodulin in Coronary Atherosclerosis Am. J. Pathol., September 1, 2001; 159(3): 797 - 802. [Abstract] [Full Text] [PDF]
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 S. N. Faust, M. Levin, O. B. Harrison, R. D. Goldin, M. S. Lockhart, S. Kondaveeti, Z. Laszik, C. T. Esmon, and R. S. Heyderman Dysfunction of Endothelial Protein C Activation in Severe Meningococcal Sepsis N. Engl. J. Med., August 9, 2001; 345(6): 408 - 416. [Abstract] [Full Text] [PDF]
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A. Y. Kim, P. L. Walinsky, F. D. Kolodgie, C. Bian, J. L. Sperry, C. B. Deming, E. A. Peck, J. G. Shake, G. B. Ang, R. H. Sohn, et al. Early Loss of Thrombomodulin Expression Impairs Vein Graft Thromboresistance: Implications for Vein Graft Failure Circ. Res., February 8, 2002; 90(2): 205 - 212. [Abstract] [Full Text] [PDF]
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), or HPC4 (
) were blocked
and then saturated with sEPCR. Biotin-PR3 was added as indicated, and
bound probe was detected with streptavidin-alkaline phosphatase and
Blue Phos substrate (absorbance at 650 nm). B,
PR3-coated wells were preincubated with increasing concentrations of
unlabeled sEPCR. Biotin-sEPCR (500 nM) was added, and bound biotin was
detected with streptavidin-alkaline phosphatase and Blue Phos
substrate.
