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Department of Internal Medicine, University of Utah Health Sciences Center, Salt Lake City, UT 84132
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptors by the Fc component of
ANCA, which is a potent stimulus for the respiratory burst and PMN
degranulation (5). The reactive oxygen species and
proteinases that are released from PMN under these conditions may
contribute to the aggressive vascular inflammation and injury that is
characteristic of Wegener’s granulomatosis.
Despite the intense interest in membrane-bound PR3 on PMN in Wegener’s
granulomatosis, remarkably little is known about the localization,
activities, and roles of PR3 in inflammatory cells from healthy
subjects. Studies of purified PR3 have shown that the soluble form of
the proteinase can degrade a number of extracellular matrix
macromolecules and heat shock proteins (6), and that it
can also cleave and activate the precursor forms of cytokines (7, 8), including IL-8, IL-1ß, and TNF-
. However, little is
known about the mechanisms by which PR3 might first come into contact
with the immune system in normal individuals. Although PR3 has been
identified on the surface of human PMN (9, 10, 11), there have
been no studies of the quantity of enzyme or its catalytic activity. In
this respect, it is noteworthy that previous work from our laboratory
has shown that activation of PMN with cytokines and chemoattractants
results in striking up-regulation of catalytically active HLE and CG on
the cell surface of PMN from healthy donors, and that membrane-bound
HLE and CG are both remarkably resistant to inhibition by physiologic
inhibitors (12, 13, 14, 15).
In this paper, we have studied bioactive forms of PR3 in PMN from healthy individuals. Our results have shown that PMN contain PR3 at mM concentrations within their azurophil granules, and that PR3 is also expressed on the cell surface of PMN. Expression of PR3 at the cell surface of PMN is rapidly and strikingly up-regulated by proinflammatory mediators, and this form of PR3 is catalytically active yet substantially resistant to inhibition by naturally occurring proteinase inhibitors. These data indicate that, when localized to the cell surface of PMN, PR3 may have important biologic activities during physiologic processes, as well as in the pathogenesis of diseases such as Wegener’s granulomatosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human serum albumin was obtained from the American Red Cross
(Washington, DC).
1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine
(platelet activating factor (PAF)) was purchased from Bachem
(Torrance, CA). TNF- was obtained from Genzyme (Boston, MA). HBSS
was purchased from Life Technologies (Grand Island, NY). Permount was
purchased from Fisher Scientific (Pittsburgh, PA). Goat anti-rabbit
IgG conjugated to rhodamine and goat anti-rabbit conjugated to HRP
were obtained from Cappel (Durham, NC). Goat anti-rabbit IgG
conjugated to colloidal gold particles was purchased from Chemicon
International (Temecula, CA). Purified human PR3, rabbit antiserum to
human PR3, and Boc-Alanine-Alanine-Norvaline-thiobenzyl ester
(Boc-Ala-Ala-Nva-SBzl) were purchased from Elastin Products
(Owensville, MO). Rabbit antiserum to HLE and control (preimmune)
rabbit serum were obtained from Zymed (San Francisco, CA). Polyclonal
rabbit anti-human CG was purchased from Dako (Carpinteria,
CA).
Z-Gly-Leu-Phe-choromethyl ketone (CG/CMK),
methoxy-succinyl-Ala-Ala-Pro-Val-7-amino-4-trifluoromethyl
coumarin, and succinyl-Ala-Ala-Phe-7-amino-4-trifluoromethyl coumarin
were purchased from Enzyme Systems Products (Livermore, CA).
1-Proteinase inhibitor
(1-PI) was obtained from Bayer (New Haven,
CT). Secretory leukocyte proteinase inhibitor (SLPI) was obtained from
Amgen (Boulder, CO). Recombinant human elafin was a generous gift from
Dr. J. M. Sallenave (Rayne Laboratory, Department of Medicine,
Edinburgh University, Scotland, U.K.). All other reagents were
purchased from Sigma (St. Louis, MO).
PMN isolation
Human PMN (>95% pure) were obtained from peripheral blood of healthy donors using the Ficoll-Hypaque technique (16). Extracts of PMN were prepared in 0.05 M phosphate buffer (pH 7.4) containing 0.04% (v/v) Triton and 1 M NaCl.
Competitive binding ELISA for PR3, HLE, and CG
Microtiter plates (Nunc, Naperville, IL) were coated with 1 µg of PR3 in 10 mM phosphate buffer containing 0.6 M NaCl (pH 7.4) overnight at 4°C, washed three times to remove unbound protein, then incubated for 1 h at 37°C with 100 ml 1% (w/v) BSA in 10 mM phosphate buffer containing 0.6 M NaCl, and 0.05% (v/v) Tween to block additional protein binding sites. PR3 standards and unknowns were incubated overnight at 4°C in a volume of 150 µl in polypropylene radioimmunoassay vials (Starstedt, Princeton, NJ) with 50 µl of rabbit antiserum to PR3 (diluted 1:1600). A total of 100 µl of each sample were transferred to the Ag-coated wells and incubated for 90 min at 4°C. The plates were washed three times and incubated for 60 min at 37°C with 100 ml HRP-conjugated goat anti-rabbit IgG diluted 1:4000. The assay was developed using o-phenylenediamine in 50 mM citrate (pH 4.5), and the reaction was stopped using 25 µl 2 N H2SO4. Absorbances were read using an automated microtiter plate reader (Molecular Devices, Palo Alto, CA). The assay was sensitive to 4 ng PR3/ml. Competitive binding ELISAs for HLE and CG were performed, as described previously (17).
PMN stimulation and fixation
PMN were resuspended at 5 x 106/ml
in HBSS containing 1 mM Ca2+ and 1 mM
Mg2+, then incubated at 37°C for 15 min with or
without PMA (50 ng/ml) or A23187 (1 µM). Cells were also incubated at
37°C for 5 min with 5 µg/ml cytochalasin B then for 10 min with
10-8 M fMLP. We also assessed the effects of
biologically relevant mediators. Cells were incubated for 30 min with
or without 10-9 M PAF, 100 U/ml TNF- , 100
ng/ml bacterial LPS from Escherichia coli 0111:B4,
10-8 M IL-8, or 10-8 M
fMLP. Cells were also primed for 15 min with
10-9 M PAF, 100 U/ml TNF-, 100 ng/ml LPS,
then activated for 30 min with 10-8 M fMLP or
10-8 M IL-8. Following activation, cells were
fixed for 3 min at 4°C with PBS containing 3% (w/v) paraformaldehyde
and 0.5% (v/v) glutaraldehyde, and then washed in HBSS. We have shown
previously that these concentrations of cytokines and chemoattractants
do not adversely affect PMN viability (12, 14).
Immunofluorescence staining
We used quantitative immunofluorescence microscopy to assess the effects of agonists on cell surface expression of PR3. Our previous work has confirmed that this technique detects serine proteinases on the cell surface of PMN, rather than intracellular enzymes (13). Fixed cells were incubated at 4°C in HBSS containing 1% (v/v) human serum albumin and 50 µg/ml goat IgG to reduce nonspecific binding of Abs. To stain for membrane-bound PR3, the PMN were incubated for 45 min at 4°C with rabbit antiserum to PR3 or control (preimmune) rabbit serum, then washed twice to remove excess Ab. Cells were then incubated for 45 min at 4°C with rhodamine-conjugated goat anti-rabbit IgG and washed twice in HBSS. Cytocentrifuge preparations were mounted in 25% (v/v) glycerol in PBS containing 250 µg/ml p-phenylenediamine to reduce photobleaching, and coverslips were applied.
The immunostained cells were examined with phase-contrast and incident-light fluorescence microscopy (Leitz Dialux 20 with L2 filter set and Leitz NPL Fluotar 40x, N.A. 1.32 objective; Leica, Deerfield, IL). Cell surface immunofluorescence was quantified by image analysis using MetaMorph software, (Universal Imaging, Westchester, PA) as described previously (13). The fluorescence intensity of 100–150 cells in each group was quantified. To correct for nonspecific staining, the mean fluorescence value for the group of cells that were incubated with the control rabbit serum was subtracted from each of the values for cells that were incubated with the rabbit antiserum to PR3, and the mean specific fluorescence value was determined. To determine the percentage of cells that express PR3, we calculated the percentage of cells that were stained with the antiserum to PR3, that had an integrated fluorescence value greater than the mean fluorescence + 3 SD of the cells that were incubated with the control rabbit serum.
Immunogold staining of PMN
Unstimulated cells, PMN activated with A23187, or PMN primed with LPS then activated with IL-8 were fixed then incubated with rabbit antiserum to PR3 or control rabbit serum, as described above. The cells were then incubated with goat anti-rabbit IgG conjugated to colloidal gold particles (diameter 20 nm) and washed twice in HBSS. Cytocentrifuge preparations were mounted in Permount, coverslips were applied, and the cells were examined by phase-contrast and polarization reflection microscopy (polarized incident light, when reflected from gold particles, passes through the cross-polarizer in the Leitz RK reflection contrast 100x, N.A. 1.32 objective).
Quantification of free release of PR3 by activated PMN
PR3, HLE, and CG were quantified in cell-free supernatant fluids from primed and stimulated PMN and in cell extracts of unstimulated PMN (prepared in HBSS containing 1 M NaCl and 0.04% (v/v)Triton) using competitive binding ELISAs, as described above.
Catalytic activity of membrane-bound PR3
To inactivate proteinases expressed endogenously on the surface of PMN, unstimulated PMN were fixed then heated to 100°C for 5 min and chilled on ice. The cells were incubated overnight at 4°C with 1 mM PMSF, then washed three times in HBSS to remove PMSF. We confirmed that the cells were intact when examined by phase contrast microscopy, and had no activity against Boc-Ala-Ala-Nva-SBzl, a synthetic substrate for PR3 (as described below), methoxy-succinyl-Ala-Ala-Pro-Val-7-amino-4-trifluoromethyl coumarin, or succinyl-Ala-Ala-Phe-7-amino-4-trifluoromethyl coumarin, synthetic fluorogenic substrates which are specific for HLE and CG, respectively, as described previously (14, 15).
The cells were then incubated overnight at 4°C with purified PR3 (1 µg/106 cells), fixed, then washed three times to remove unbound enzyme. We have shown previously that our fixation process does not affect the catalytic activity of membrane-bound serine proteinases (13, 15). Membrane-bound PR3 activity was assayed using Boc-Ala-Ala-Nva-SBzl as the substrate (18). Briefly, 106 PMN or 3.1–80 ng soluble PR3 in a volume of 100 ml HBSS were added to 900 ml of 0.5 M NaCl containing 0.1 M HEPES and 400 µM 4',4'-dithiodipyridine and 100 µM Boc-Ala-Ala-Nva-SBzl (pH 7.5). The samples were incubated for 2 h at 37°C, then the absorbances of cell-free supernatant fluids were determined at 324 nM using a DU8 spectrophotometer (Beckman Instruments, Palo Alto, CA).
To assess whether membrane-bound PR3 is active against soluble human fibronectin, exogenous PR3 bound to fixed PMN, fixed PMN incubated without PR3 (both at 2.5 x 106/assay), or soluble PR3 (125 ng/assay) were incubated overnight in 50 µl HBSS with 25 µg soluble human fibronectin. To confirm that there was no detachment of PR3 from the surface of PMN, we incubated PMN that bound exogenous PR3 overnight at 37°C in HBSS alone, then harvested the cell-free supernatant fluid, and incubated the latter with soluble human fibronectin, as described above. Cell-free supernatant fluids were reduced with 10% (v/v) ß-mercaptoethanol, then were subjected to 7.5–15% gradient SDS-PAGE (13). To provide further assurance that the fibronectin-degrading activity associated with the cells was due to membrane-bound PR3, we incubated soluble PR3 (125 ng/assay) or exogenous PR3 that was bound and fixed onto the cell surface of PMN both in the presence and absence of 2 µM elafin or SLPI in a total volume of 50 µl HBSS for 15 min at 25°C. The samples were then incubated with soluble human fibronectin, and cell-free supernatant fluids were analyzed by SDS-PAGE, as described above.
Susceptibility of membrane-bound PR3 to inhibition
Purified PR3 (25 ng) or exogenous PR3, which was bound and fixed
onto the cell surface of PMN (106/assay), were
incubated at 25°C for 15 min in an assay volume of 100 ml HBSS with
or without the following inhibitors: 1) 5 µM
1-PI, 2) 2 µM SLPI, 3) 2 µM elafin, 4) 1
mM PMSF, 5) 100 µM CG/CMK, or 6) 2 µM
1-antichymotrypsin. Residual PR3 activity was
quantified in cell-free supernatant samples using Boc-Ala-Ala-Nva-SBzl,
as described above. The results were expressed as percent
inhibition.
Elution of PR3, HLE, and CG from the cell surface of activated PMN with increasing ionic strength
PMN were optimally primed with PAF and stimulated with fMLP, then fixed (to prevent leakage of intracellular proteinases; Refs. 12 and 14), as described above. Cells were resuspended at 6 x 106/ml in PBS containing 0.15, 0.3, 0.5, 0.75, 1.0, and 1.25 M NaCl. The cells were incubated overnight at 4°C on an orbital rotator. Cells were also resuspended in PBS containing 0.15 M NaCl, and incubated at 37°C for up to 60 min. Cell-free supernatant fluids and cell extracts of unstimulated PMN were assayed for immunoreactive PR3, HLE, and CG using competitive binding ELISAs, as described above.
Statistics
Data are expressed as mean ± SEM or mean ± SD. The results for paired and unpaired data were compared using the Student t test for parametric data and the Mann-Whitney rank sum test for nonparametric data; p values <0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We found that PMN contain 3.24 ± SD 0.24 µg of PR3 per 106 cells (or 3.24 pg per cell; n = 17 donors). Assuming that HLE and PR3 reside in the same azurophil granules, we can use our data together with the mean number of HLE-containing azurophil granules per PMN (399 ± (SEM) 20; Ref. 19), and the mean volume of a single azurophil granule (2.09 x 10-17 L; Ref. 20), to calculate that, on average, each azurophil granule contains 8.12 fg (168,644 molecules) of PR3 at a mean concentration of 13.4 mM.
Cell surface expression of PR3 in response to pharmacologic agonists
We first tested the effects of pharmacologic agonists that are
potent inducers of PMN degranulation (PMA, cytochalasin B and fMLP, and
the calcium ionophore, A23187). Unstimulated cells had low, but
detectable levels of PR3 on their cell surface (Fig. 1
A). Activation of cells with
all of the pharmacologic agents tested induced highly significant
increases in cell surface expression of PR3. A23187 was consistently
the most potent inducer of the expression of PR3 on the surface of PMN,
inducing 
10-fold increases in expression of PR3 when compared with
unstimulated cells.
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Activation of cells with optimal concentrations of PAF, TNF-,
LPS, or IL-8 alone induced modest 1.5- to 2.5-fold increases in cell
surface expression of PR3 on PMN (Fig. 1
B). However,
synergistic increases in cell surface expression of PR3 were observed
when cells were first primed for 5–15 min with LPS, PAF, or TNF-,
then activated for 30 min with IL-8 (conditions that induce optimal
expression of HLE and CG on the surface of PMN (12, 14)).
Primed and activated PMN expressed 5- to 6-fold more PR3 on their cell
surface when compared with unstimulated cells. Similar results were
obtained when cells were primed with cytokines, then activated with
fMLP (data not shown). The priming effects of cytokines were rapid and
persistent. Following priming of cells with TNF-, significant
increases in cell surface expression of PR3 were observed within 1 min,
and persisted for at least 90 min after exposure to IL-8 (data not
shown).
In addition to increasing the mean quantity of PR3 on the cell surface
of PMN, priming of cells with cytokines followed by activation of cells
with IL-8 also increased the percentage of cells that express PR3. We
found that 38.0 ± (SEM) 13.7% (9.5% to 85.0%) of unstimulated
cells expressed PR3 compared with 65.1 ± 9.1%
(p = 0.062), 70.7 ± 11.4%
(p = 0.014), and 78.1 ± 6.9%
(p = 0.008) of cells primed with LPS, PAF, and
TNF-, respectively, then activated with IL-8 (n = 5
donors). Together, these data indicate that exposure of cells to
proinflammatory mediators induces rapid and persistent increases in the
mean quantity of PR3 on the cell surface of PMN as well as significant
increases in the percentage of cells that express PR3 on their cell
surface.
Immunogold localization of PR3 on the cell surface of activated PMN
To exclude the possibility that our immunofluorescence technique
was detecting intracellular PR3, we localized PR3 immunoreactivity
using a second Ab conjugated to colloidal gold particles with a
diameter of 20 nm (which are too large to penetrate cells). There were
a few gold particles associated with unstimulated cells that were
stained for cell surface-bound PR3 (Fig. 2). In marked contrast, cells that were
activated with either A23187 or LPS and IL-8, then stained for PR3 had
numerous gold particles localized to their cell surface. There was
almost a complete lack of gold particles associated with LPS and IL-8
activated cells (Fig. 2) or A23187-activated cells (not shown) that
were incubated with the control rabbit serum. These data confirm that
PR3 translocates to the external surface of the plasma membrane of PMN
following cellular activation.
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To quantify free release of PR3 in response to cellular
activation, PMN were optimally primed with cytokines and activated with
IL-8. Immunoreactive PR3, HLE, and CG were then quantified in cell-free
supernatant fluids and also in cell extracts of unstimulated PMN using
competitive binding ELISAs. Only 2–3% of the cellular content of
PR3 in unstimulated cells was released from PMN during activation, and
this was similar to the quantities of HLE and CG that were freely
released from cells under the same conditions (Table I).
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To provide further assurance that PR3 can bind to the external
surface of the plasma membrane of PMN, we tested whether exogenous PR3
can bind to viable PMN. Cells were incubated at 4°C with or without
varying concentrations of soluble PR3, then cell surface-bound PR3 was
quantified using immunofluorescence staining and image analysis. Cells
that were exposed to exogenous PR3 expressed significantly more cell
surface-bound PR3 when compared with cells incubated without PR3, and
exogenous PR3 bound to viable PMN in a dose-dependent manner (Fig. 3). These data confirm that PR3 can bind
to sites on the external surface of the plasma membrane of
PMN.
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We assessed whether PR3 on the surface of PMN is catalytically active against Boc-Ala-Ala-Nva-SBzl, a synthetic substrate that is cleaved by PR3. For these studies, it was not possible to study PR3 that is endogenously expressed on cells following activation because HLE and CG are also expressed on the surface of activated PMN (12, 13, 14), and there is no substrate available that is specific for PR3 activity. To circumvent this problem, we studied the catalytic activity of exogenous PR3 bound and fixed onto unstimulated cells, as described in Materials and Methods.
Fixed PMN that were not exposed to PR3 had no detectable activity against Boc-Ala-Ala-Nva-SBzl (a chromogenic substrate which is cleaved by PR3), or against methoxy-succinyl-Ala-Ala-Pro-Val-7-amino-4-trifluoromethyl coumarin or succinyl-Ala-Ala-Phe-7-amino-4-trifluoromethyl coumarin (fluorogenic substrates that are specific for HLE and CG, respectively). Cells that bound exogenous PR3 had activity equivalent to 45.5 ± (SD) 13.9 ng of soluble PR3 per 106 cells (n = 8 experiments). These data indicate that cell surface-bound PR3 on PMN is catalytically active against an oligopeptide substrate.
Next, we tested whether cell surface-bound PR3 on PMN can degrade human
fibronectin, a component of the extracellular matrix and basement
membranes. We incubated the following with soluble human fibronectin
for 3 h: 1) exogenous PR3 bound and fixed onto PMN; 2) fixed PMN
that were incubated without exogenous PR3 (as a control); or 3) soluble
PR3. Cell-free supernatant fluids were then reduced and subjected to
SDS-PAGE. Soluble PR3 and exogenous PR3 bound to PMN completely or
substantially degraded the fibronectin substrate (Fig. 4, A and B). In
marked contrast, there was no degradation of fibronectin that was
incubated with the control PMN that were not exposed to PR3 (Fig. 4A). When cell-free supernatant fluids were harvested from
the PMN that bound exogenous PR3, then incubated with fibronectin under
the same conditions as the cells, no degradation of the fibronectin was
observed (Fig. 4A). Together, these data indicate that fixed
PMN that bind exogenous PR3 to their cell surface can degrade
fibronectin, and that this activity is mediated by cell surface-bound
PR3, and is not due to leakage of intracellular proteinases, or
detachment of PR3 from the surface of the fixed PMN.
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B). In contrast,
SLPI was ineffective against either soluble or membrane-bound PR3.
Together, these data confirm that membrane-bound PR3 on PMN degrades
fibronectin. Membrane-bound PR3 is resistant to inhibition by high molecular mass inhibitors
We compared the susceptibility of soluble vs membrane-bound PR3 to
inhibition by proteinase inhibitors that vary in their molecular size
using Boc-Ala-Ala-Nva-SBzl as the substrate. All of the inhibitors of
PR3 that were tested (PMSF, elafin, and 1-PI)
were effective inhibitors of soluble PR3 (Fig. 5). As expected, SLPI did not inhibit
soluble PR3. PMSF, a low molecular mass inhibitor of serine proteinases
(Mr = 174 Da), was almost fully
effective against membrane-bound PR3. In contrast, the naturally
occurring inhibitors elafin (Mr = 6
kDa) and 1-PI
(Mr = 52 kDa) were only partially
effective against membrane-bound PR3 despite the fact that these
inhibitors were used in the assay at a 113 ± SD 25.4-fold and a
282.9 ± 63.5-fold molar excess over membrane-bound PR3,
respectively. As controls, we also tested CG/CMK and
1-antichymotrypsin (which are low and high
molecular mass inhibitors of CG, respectively). As expected, the
inhibitors of CG were ineffective against both soluble and
membrane-bound PR3. These data indicate that membrane-bound PR3 on PMN
is substantially resistant to inhibition by physiologic inhibitors
of PR3.
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We assessed the ability of solutions with increasing ionic
strength to elute PR3 from the cell surface of PMN that had been
optimally activated to induce cell surface expression of PR3, then
fixed to prevent release of intracellular proteinases (12, 14). We compared elution of PR3 to that of HLE and CG, which are
both more highly positively charged than PR3 and are known to bind to
the PMN cell membrane via ionic interactions (12, 14, 23).
Increasing the ionic strength of the solution to 0.75 M NaCl resulted
in maximal elution of PR3 from the cell surface of PMN (Fig. 6A). As expected, HLE required
higher concentrations of NaCl (up to 1 M) for maximal elution, and
elution of CG (which is the most cationic of the three proteinases) was
not complete even when cells were exposed to 1.25 M NaCl. These results
did not represent leakage of intracellular enzyme into supernatant
fluids, because less than 0.4% of the PMN cellular content of lactate
dehydrogenase activity is detected in cell-free supernatant fluids when
cells are incubated under these conditions (12, 14).
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A is that exposure of PMN to 0.15 M
NaCl resulted in significantly greater elution of PR3 from the cell
surface of PMN when compared with elution of HLE
(p = 0.01) or CG (p =
0.007). To test the release of these enzymes from the PMN cell surface
under physiologic conditions, we directly compared the release of PR3,
HLE, and CG from the cell surface of activated PMN in the presence of
0.15 M NaCl at 37°C, as a function of time (Fig. 6B).
There was significantly greater release of PR3 from the surface of
activated PMN when compared with release of HLE and CG at all time
points tested. Release of PR3 was detected within 5 min of exposure to
isotonic buffer, and was almost complete by 15 min. In marked contrast,
there was minimal release of either HLE and almost no release of CG
from the surface of PMN in the presence of physiologic salt
concentrations, even when the incubation period was extended to 2
h (data not shown). These data indicate that during the inflammatory
response, a portion of cell surface-bound PR3 is rapidly released from
the plasma membrane of activated PMN, whereas HLE and CG remain bound
to the cell surface. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Quantification of PR3 in PMN
Our results demonstrated that PMN contain 3 pg of PR3 per cell,
which is in good agreement with recently published data from
Witko-Sarsat et al. (24), and it is also very similar to
the quantity of HLE (1.1 pg per cell) and CG (0.85 pg per cell)
contained within PMN (17). From our data, together with
previously published data on the mean number of HLE-containing
azurophil granules per PMN (19), and the mean volume of a
single azurophil granule (20), we have calculated that
each granule contains PR3 at a mean concentration of 13.4 mM.
Regulation of the expression of cell surface-bound PR3 activity
We used quantitative immunofluorescence staining to study PR3
expression on the surface of PMN, and immunogold staining confirmed
that our Ab recognizes cell surface-bound PR3 rather than intracellular
proteins. We have shown that, on average, 38% of unstimulated PMN
from healthy donors express low levels of PR3 on their cell surface,
and that cell surface expression of PR3 on PMN is readily inducible
both by pharmacologic agonists and proinflammatory mediators with
respect to both the mean quantity of PR3 expressed per cell and the
percentage of cells that express PR3. The relatively greater effects of
pharmacologic agonists on mean cell surface expression of PR3, when
compared with the effects of cytokines and chemoattractants when used
alone, are likely to reflect their greater effects on PMN
degranulation.
Other laboratories have shown a proportion of unstimulated PMN from
healthy donors express PR3 on their cell surface. Halbwachs-Mercarelli
et al. (11) demonstrated that the proportion of freshly
isolated PMN that expresses PR3 varies considerably between donors
(mean 49.6%; range 0–95%; n = 47 donors), but is
extremely stable for each individual over prolonged periods of time.
Although we did not study our donors over time, we also found a wide
range in the proportion of freshly isolated PMN from healthy
individuals that express low levels of PR3 (9.5–85%). Other
laboratories have shown that exposure of cells to agonists such as PMA,
TNF-, IL-8, fMLP, cytochalasin B, and TGF-ß increases the
proportion of PMN that express PR3 on their cell surface (2, 9, 10, 11), but the effect of these mediators on the mean quantity
of cell surface-bound PR3 on the whole PMN population has not been
assessed previously. It is noteworthy that one of these studies found
that 70% of PMN from patients with sepsis syndrome express PR3 on
their cell surface (9). Our data suggest that exposure of
PMN to cytokines and chemoattractants in vivo could mediate the
observed increases in cell surface expression of PR3 on PMN during
sepsis.
Although priming of cells with cytokines, followed by activation with a chemoattractant, results in striking synergistic increases in cell surface expression of PR3, minimal PR3 is freely released from cells even under these conditions. These data indicate that when cells are activated by exposure to proinflammatory mediators in vivo, PR3 bound to the cell surface of PMN is likely to be the predominant extracellular form of the enzyme.
Catalytic activity and susceptibility to inhibition of membrane-bound PR3
We are the first to report that membrane-bound PR3 on PMN is
catalytically active. Earlier studies were hampered by the lack of
availability of a substrate that is completely specific for PR3. The
catalytic activity of endogenously expressed PR3 on the cell surface of
activated PMN cannot be studied directly because activated PMN also
express cell surface-bound HLE (13, 14), which has a
substrate specificity that is similar to that of membrane-bound PR3. To
circumvent this problem, we studied exogenous PR3 bound to
unstimulated, fixed PMN. Cells that bound exogenous PR3 cleaved a
synthetic oligopeptide substrate. Although it is not possible to use
this substrate to quantify catalytically active PR3 that is
endogenously expressed on the surface of activated PMN, it is
noteworthy that optimally activated PMN express 12% of their
cellular content of both HLE and CG on their cell surface (12, 14), and it is likely that a similar proportion of the PMN
cellular content of PR3 is also expressed on the surface of optimally
activated cells.
We also demonstrated that exogenous PR3 bound to PMN can degrade fibronectin, an important component of the extracellular matrix and basement membranes. These data suggest that PR3 expressed on the surface of activated PMN may play roles in the egress of cells from the vasculature and their penetration of tissues barriers, including basement membranes, as cells traverse through tissue planes en route to sites of inflammation.
We are also the first to study the susceptibility of membrane-bound PR3
on PMN to inhibition by proteinase inhibitors that vary in their
molecular size. In marked contrast to soluble PR3, membrane-bound PR3
was substantially resistant to inhibition by physiologic inhibitors
including 1-PI, which is the major inhibitor
of PR3 in plasma and the lower respiratory tract (25), and
elafin, which may be an important inhibitor of PR3 in various
secretions (26), even when these inhibitors were used at
100- to 300-fold molar excess over enzyme. The fact that the low
molecular mass inhibitor, PMSF, was almost fully effective against
membrane-bound PR3 indicates that steric hindrance might be the major
mechanism by which membrane-bound PR3 evades inhibition by intermediate
and high molecular mass inhibitors.
Mechanism of binding of HLE to the cell surface of PMN
Previous enzyme histochemical studies have demonstrated that, following stimulation of PMN, azurophil granules translocate to the cell surface and that serine proteinases become bound to the external surface of the plasma membrane near the sites of degranulation (13). Previous studies of HLE and CG have shown that the strongly cationic nature of HLE and CG enables them to bind to a negatively charged plasma membrane constituent or constituents (12, 14, 23). We tested the possibility that PR3 also binds to the cell surface by a charge-dependent mechanism, because PR3 is also a cationic proteinase (27). Our results demonstrated that elution of PR3 from the cell surface of PMN was detectable even in the presence of physiologic NaCl concentrations, and that PR3 eluted in solutions having lower ionic strength when compared with the elution of HLE, which in turn, eluted in solutions having lower ionic strength when compared with elution of CG. These data are entirely consistent with our working hypothesis, because PR3 is the least cationic, and CG is the most cationic of the three serine proteinases (isoelectric points for PR3, HLE, and CG are 9.1, 10.5, and >11, respectively (27, 28)). Detachment of PR3 from the cell surface of activated PMN is also likely to occur at sites of inflammation in vivo, and this phenomenon could be relevant to the pathogenesis of systemic necrotizing vasculitides.
We disagree with the previous findings of Witko-Sarsat et al. (2), who reported that PR3 cannot be eluted from the cell surface of human PMN. This discrepancy is likely to reflect two major technical differences between the two studies. First, Witko-Sarsat et al. studied elution of PR3 from the cell surface of unstimulated PMN, whereas we studied elution of PR3 from cells that had been activated to induce cell surface expression of PR3 on PMN following degranulation. It is possible that PR3 that is constitutively present on the cell membrane of unstimulated PMN differs in its mechanism of association with the plasma membrane when compared with PR3 which binds to the cell surface following cellular activation and degranulation. We consider this to be only a formal possibility. The second and more likely explanation for the discrepant results is that Witko-Sarsat and coworkers tested whether PR3 could be eluted from the cell surface of unstimulated PMN by exposing viable cells to nonphysiologic buffers having extreme pH (pH 3.0 and pH 10.7) and demonstrated small increases in cell surface expression of PR3. It is very likely that exposing PMN to extremes of pH adversely affects cell viability and membrane permeability. Any release of PR3 from cells stressed in this manner would mask reductions in cell surface expression of PR3 induced by exposure of PMN to highly acidic or basic pH. Witko-Sarsat and coworkers did not test the effects of their treatments on cell viability or free release of PR3, nor did they study the effects of extreme pH on elution of HLE and CG (as controls), which have been shown to bind via ionic interactions to the cell membrane of PMN (12, 14, 25). In the current study, we exposed activated PMN to solutions having increasing ionic strength after fixing the cells to prevent release of intracellular proteinases. With this experimental design, we were able to demonstrate elution of PR3, HLE, and CG from the cell surface in an order that was related to their isoelectric points. We have also confirmed that there is no significant release of LDH activity from PMN during this assay. We are thus confident that PR3 can be eluted from the cell surface of PMN (and that it is released from the cell surface even under physiologic conditions) and that ionic interactions are important in the binding of this proteinase to the cell membrane following cellular activation.
Our data indicate that the binding of serine proteinases to the cell membrane may be a mechanism by which cells preserve and restrict the catalytic activity of their serine proteinases to the pericellular environment, and thereby ensure controlled extracellular proteolysis during physiologic processes. A potentially important difference between PR3 and the other two serine proteinases, however, is that PR3 detaches more readily from the surface of PMN when compared with HLE or CG, and PR3 could thus become exposed to immune-competent cells that are some distance removed. This process might lead to the greater propensity for genetically susceptible individuals to generate ANCA directed against PR3, as opposed to HLE or CG.
The molecule(s) on the PMN plasma membrane to which PR3 binds remain unknown. However, ongoing studies in our laboratory indicate that PMN express common, low affinity, yet high-volume binding sites for HLE and CG (C.A.O. and E.J.C., unpublished observations). In addition, treatment of PMN with chondroitinase partially inhibits the binding of HLE and CG to PMN indicating that binding of these serine proteinases to PMN is mediated, in part, by chondroitin sulfate-containing proteoglycans. We have also shown that excess unlabeled PR3 can compete with labeled HLE or CG for binding to the plasma membrane indicating that PR3 binds to sites with similar characteristics (C.A.O. and E.J.C., unpublished observations). Low affinity, high-volume binding sites would be ideally suited for binding the mM concentrations of PR3 that are transiently present near the cell surface of PMN following degranulation. Other biologically important mediators have also been shown to bind to negatively charged proteoglycans on cell surfaces. For example, platelet factor 4 has been shown to bind to chondroitin sulfate proteoglycans on the cell surface of PMN (29). In addition, heparan sulfate proteoglycans on the cell surface of endothelial cells and fibroblasts serve as coreceptors for basic fibroblast growth factor, which protect basic fibroblast growth factor from proteolytic degradation (30, 31, 32). Cell surface proteoglycans may thus serve to focus, regulate and preserve the activity of diverse bioactive molecules on inflammatory cells and resident cells in vivo.
Conclusions
PR3 that is bound to the cell surface of PMN retains its activity
even in the presence of physiologic inhibitors, and may thereby play
important roles in the physiologic processes of PMN. The binding of PR3
to the surface of inflammatory cells serves to focus, preserve, and
restrict the catalytic activity of this proteinase to the immediate
pericellular environment. Because membrane-bound PR3 can degrade large
polypeptide substrates, such as fibronectin, this form of the
proteinase may play a role in the penetration of tissue barriers by PMN
as they migrate through tissues. Moreover, the observations that
soluble PR3 can also activate precursor forms of cytokines such as
TNF-, IL-8, and IL-1ß (7, 8) suggest the possibility
that catalytically active PR3 expressed on the cell surface of PMN may
also participate in cytokine processing, and thereby play a role in
regulating inflammation.
Rapidly inducible expression of PR3 on the cell surface of PMN in healthy individuals may be a normal phenomenon during the inflammatory response, and cell surface-bound PR3 may be the predominant extracellular form of the proteinase when cells are activated by proinflammatory mediators. However, in susceptible individuals, persistent expression of PR3 on the cell surface of inflammatory cells and/or detachment of PR3 from the surface of activated cells may lead to the generation of autoantibodies, which in turn activate more PMN and contribute to self-perpetuating tissue injury in systemic necrotizing vasculitis syndromes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Caroline A. Owen, Department of Internal Medicine, University of Utah Health Sciences Center, 410 Chipeta Way, Room 108, Salt Lake City, UT 84108.
3 Abbreviations used in this paper: PR3, proteinase 3; 1-PI, 1-proteinase inhibitor; ANCA, anti-neutrophil cytoplasmic Abs; CG, cathepsin G; HLE, human leukocyte elastase; PAF, platelet activating factor; PMN, polymorphonuclear neutrophils; SLPI, secretory leukocyte proteinase inhibitor; Boc-Ala-Ala-Nva-SBzl, Boc-Alanine-Alanine-Norvaline-thiobenzyl ester; CG/CMK, Z-Gly-Leu-Phe-choromethyl ketone.
Received for publication March 9, 2000. Accepted for publication June 28, 2000.
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1-Antitrypsin: molecular pathology, leukocytes, and tissue damage. J. Clin. Invest. 78:1427.
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) were incubated for 30 min with or without optimal
concentrations of TNF-
), and CG (
). Note that elution of
PR3 was detectable in the presence of 0.15 M NaCl, and was maximal in
the presence of 0.75 M NaCl. Higher concentrations of NaCl were
required to elute HLE and CG from the cell surface of PMN. Data are
mean values; error bars represent SEM; n = 3
donors. *, p < 0.05; **,
p < 0.01 compared with elution of the same
proteinase in the presence of 0.15 M NaCl. B, PMN were
resuspended in PBS containing 0.15 M NaCl, incubated at 37°C for
varying times, then immunoreactive PR3 (•), HLE (