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Infect Immun, August 1998, p. 3611-3617, Vol. 66, No. 8
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Role of Protegrins and Other Elastase-Activated
Polypeptides in the Bactericidal Properties of Porcine
Inflammatory Fluids
Jishu
Shi and
Tomas
Ganz*
Will Rogers Institute Pulmonary Research
Laboratory, Departments of Medicine and Pathology, School of
Medicine, University of California at Los Angeles, Los Angeles,
California 90095
Received 19 February 1998/Returned for modification 2 April
1998/Accepted 21 May 1998
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ABSTRACT |
The mammalian host response to infection includes the production
and secretion of antimicrobial peptides from phagocytes and epithelial
cells. Protegrins, a group of broadly microbicidal peptides isolated
originally from porcine neutrophil lysates, were found to be stored as
inactive proforms in porcine neutrophil granules but could be activated
extracellularly by neutrophil elastase. We assessed the biological role
of protegrins and other elastase-activated polypeptides in the
microbicidal activity of neutrophil secretions and inflammatory fluids.
When stimulated with phorbol myristate acetate (PMA), neutrophils
generated stable microbicidal activity against both Escherichia
coli and Listeria monocytogenes under normal-salt
conditions and in the presence of 0 to 10% serum. The generation of
these antimicrobial substances was dependent on neutrophil elastase,
since it was inhibited by 1 mM
N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone when it was present during activation, but not when this inhibitor was added
afterwards. However, elastase-dependent activation of proprotegrins to
protegrins in PMA-stimulated neutrophils was not inhibited by the
presence of 1 to 2% serum. Porcine neutrophils also released
antibacterial activity during phagocytosis of latex beads, and this too
was dependent in large part on elastase-activated polypeptides,
including protegrins. Moreover, protegrins were found at bactericidal
concentrations in cell-free abscess fluid from naturally infected pigs.
Taken together, these studies show that protegrins and other
elastase-activated polypeptides are important stable antibacterial
factors in porcine neutrophil secretions. The potential host defense
role of elastase as an activating enzyme for the precursors of
microbicidal peptides must be taken into account when therapeutic
inhibitors of neutrophil elastase are evaluated for clinical use as
anti-inflammatory agents.
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INTRODUCTION |
The host defense armamentarium of
mammals includes various antimicrobial peptides and proteins released
from phagocytes and epithelia (4). In addition to
larger antimicrobial proteins that function as enzymes (e.g.,
lysozyme or phospholipase A2) or contain binding sites for specific
microbial macromolecules (e.g., the bactericidal-permeability-inducing
protein [BPI]), mammalian phagocytes and epithelia produce two
families of small microbicidal peptides, defensins and
cathelicidins. Unlike defensins, which share a three-dimensional
structure, cathelicidins are defined by a common N-terminal
precursor motif, cathelin, joined to a highly variable C-terminal
domain that appears to be the active microbicidal moiety. Many, but not
all, cathelicidins contain a characteristic elastase cleavage site
between the anionic cathelin domain and the cationic C-terminal peptide
(28). Proteolytic processing at this site has been observed
in activated bovine (29) and porcine (16)
neutrophils and was required for microbicidal activity. In contrast,
the rabbit cathelicidins p15a and p15b lack this consensus site, their
cathelin domains are less anionic, and they are microbicidal without
proteolytic processing (26). Cathelicidins are located in
the types of neutrophil granules that are readily released into the
extracellular fluid, i.e., the specific (secondary) granules in
human and murine neutrophils and the large granules in bovine
neutrophils (3, 12, 20, 22, 29), suggesting that the
peptides may function primarily in extracellular spaces.
Neutrophils engaged in host defense also release a variety of important
short-lived microbicidal substances, such as reactive oxygen and
nitrogen intermediates, but in this study we focused on secretions that
remain stable in inflammatory fluids over a period of days. This stable
material will be referred to as neutrophil secretion. We hypothesized
that elastase-activated cathelicidins contribute substantially to the
extracellular microbicidal activity of neutrophil secretions and
inflammatory fluids. We have chosen the pig as an experimental model,
since porcine neutrophils lack defensins (9) but are
unusually well endowed with cathelicidins, including protegrins
(8), prophenins (5), PR-39 (19), and porcine myeloid antimicrobial peptides (PMAPs)
(24). The precursors for all these peptides contain a
characteristic elastase cleavage site, but, with the
exception of protegrins, their processing by elastase has not yet
been demonstrated. Of the porcine cathelicidins, the
protegrins PG1, PG2, and PG3 are among the most active and abundant (9). Their amino acid sequences have been
determined to be RGGRLCYCRRRFCVCVGRam,
RGGRLCYCRRRFCICVam, and RGGGLCYCRRRFCVCVGRam,
re- spectively (boldfaced residues differ among the three
peptides, and "am" stands for C-terminal amidation). We
previously showed that porcine neutrophils, when stimulated
with phorbol myristate acetate (PMA), generated extracellular
microbicidal activity against Listeria monocytogenes by
elastase-mediated activation of secreted proprotegrins (pPGs)
(16). In the present study, we assess the biological role of
protegrins and other elastase-activated polypeptides in the
antibacterial activity of neutrophil secretions and inflammatory fluids.
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MATERIALS AND METHODS |
Neutrophil secretions.
Porcine peripheral-blood neutrophils
were isolated from 8- to 10-week-old healthy pigs as described earlier
(18). Briefly, 30 ml of EDTA-treated blood (final
concentration, 5 mM EDTA) was mixed with 30 ml of 3% dextran in
phosphate-buffered saline (PBS) and was incubated for 30 min at room
temperature. The supernatant was carefully removed from the
dextran-sedimented blood, overlaid on 10 ml of Histopaque (Sigma), and
centrifuged at 300 × g for 25 min at room temperature.
Neutrophils were collected from the bottom of the tube, and
contaminating red blood cells were lysed with 10 ml of 0.2% NaCl for
30 s. Isotonicity was restored with 10 ml of 1.6% NaCl.
Neutrophils (>95% of the cells) were washed once in ice-cold PBS
solution without divalent cations and kept on ice.
For degranulation experiments, freshly isolated neutrophils were
resuspended at 108/ml in PBS containing 1 mM
Ca2+ and 1 mM Mg2+, with or without serum.
Neutrophils were then incubated with PMA (100 ng/ml) at 37°C for 30 min in the presence or absence of a specific inhibitor for neutrophil
elastase, N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl
ketone (CMK) (1 mM; Sigma). Neutrophil secretions were collected as
supernatant after the removal of neutrophils by centrifugation at
13,000 × g for 1 min.
For phagocytosis experiments, zymosan (Sigma) and latex beads (Difco)
were opsonized as described earlier (
6,
7). Briefly,
zymosan
(10
9 particles/ml in PBS) was tumbled with the same volume
of freshly
prepared porcine serum from a 6- to 8-week-old healthy pig
at
37°C for 30 min, then washed twice with PBS, and resuspended in
PBS at 10
9 particles/ml. Latex beads were opsonized by
mixing 0.25 ml of
Difco 0.81 latex beads, 0.05 ml of porcine serum, and
0.4 ml of
PBS and incubating at 37°C for 30 min; then they were
washed twice
with PBS and resuspended in PBS at 10
9
particles/ml. Freshly isolated neutrophils were incubated with
opsonized zymosan or latex beads (cell/particle ratio, 1:10 for
both
particles) in the presence or absence of 1 mM CMK or 0.5
mM
diisopropylfluorophosphate (DFP; Sigma), a cell-permeant serine
protease inhibitor, at 37°C for 30 min. The association of phagocytes
with particles was verified by light microscopy. Neutrophil secretions
induced by zymosan phagocytosis were collected after 1 min of
centrifugation at 13,000 ×
g to remove both
neutrophils and zymosan
particles. In the phagocytosis experiments with
latex beads, neutrophils
were collected by centrifugation at 200 ×
g for 5 min and latex
beads were then collected by
centrifugation at 13,000 ×
g for
1 min, leaving the
neutrophil secretions as supernatant.
Collection and analysis of abscess fluids.
Two otherwise
healthy pigs with abscesses (one subcutaneous, the other in the
abdominal wall) were identified in the slaughterhouse. Both abscesses
were collected and centrifuged immediately at 13,000 × g for 10 min to remove cells and cell debris. The
supernatants (abscess fluids) were subjected to a radial diffusion
microbicidal assay, then analyzed by acid-urea-polyacrylamide gel
electrophoresis (AU-PAGE), gel-overlay bactericidal assay, and Western
blot analysis with anti-PG3 antibody as described below.
Antibacterial assays. (i) Bacteria.
L. monocytogenes
EGD and Escherichia coli ML35p were used in the
antibacterial assays. Overnight cultures of bacteria in 3% Trypticase
soy broth (TSB) were subcultured for 2.5 h to exponential-growth phase in a shaking water bath at 37°C. Bacterial concentrations were
estimated photometrically (an optical density of 1 at 620 nm
corresponds to ~2.5 × 108 bacteria/ml). Bacteria
were washed and diluted with PBS to the desired concentrations.
(ii) Radial diffusion assay.
The radial diffusion assay was
performed as described previously (21). Briefly, the
underlay consisted of 1% agarose and 0.03% TSB in 10 mM sodium
phosphate with 100 mM NaCl (normal salt medium), pH 7.4. The overlay
consisted of 6% TSB and 1% agarose in PBS for all assays. Bacteria
(5 × 106) were mixed with 10 ml of underlay gel
solution (43°C) and poured into 10- by 10-cm petri dishes. A series
of wells (diameter, 3 mm) were made after the agarose solidified.
Peptide solutions or neutrophil secretions (5 µl per well) were added
to designated wells. Plates were incubated at 37°C for 3 h. The
bacterial agars were then covered with 10 ml of overlay. After 18 h of incubation at 37°C to allow visible bacterial growth, the plates
were stained with 0.001% Coomassie blue for 10 h. Antibacterial
activity was indicated by the clear zone (no bacterial growth) around
the well. The activity was represented in radial diffusion units,
defined as (diameter of clear zone in millimeters, 
3.0 mm) × 10. All assays were performed in duplicate and repeated at least once.
(iii) CFU assay.
The CFU assay was performed as described
earlier (16). Briefly, 25 µl of neutrophil secretion and 2 µl of bacterial solution (108 CFU/ml) were mixed and
incubated at 37°C in a shaking water bath for 30 min and then were
diluted 500-fold in PBS, and aliquots were plated in triplicate on
Trypticase soy agar plates. After incubation for 16 h at 37°C,
the colonies were counted and the numbers of CFU per milliliter were
calculated.
(iv) Gel overlay assay.
The gel-overlay assay was performed
as described previously (16). Briefly, proteins and peptides
were separated by AU-PAGE, and the gel was neutralized by washing for
15 min in 0.01 M PBS (pH 7.4) with 0.01 N NaOH, then in 0.01 M PBS only
for 15 min. The gel was then placed on a premade 1% agarose plate
containing 10 ml of 10 mM sodium phosphate, 100 mM NaCl, 0.1% TSB, 1%
porcine serum, and 106 L. monocytogenes or
E. coli organisms and was incubated at 37°C for 3 h
to allow the proteins and peptides in the polyacrylamide gel to diffuse
into the bacterial layer. The polyacrylamide gel was then removed, and
the bacterial layer was overlaid with a nutrient layer that contained
6% TSB in 1% agarose. After 18 h of incubation at 37°C to
allow visible bacterial growth, the agarose plate was stained with
0.001% Coomassie blue for 10 h. Antibacterial activity was
indicated by the clear zone (no bacterial growth).
ELISA for pPGs and Western blot analysis for protegrins.
For
the enzyme-linked immunosorbent assay (ELISA), 96-well plates (Becton
Dickinson Labware, Oxnard, Calif.) were coated with 1 µg of
monoclonal anti-PG3 antibody/ml (16) at 37°C for 2 h. Plates were then washed and blocked with 0.1% fetal bovine serum at
37°C for 30 min; then they were washed and incubated with 100 µl of
pPG3 (0.02, 0.04, 0.08, 0.16, 0.32, 0.64, or 0.128 µg/ml) or
neutrophil secretion samples (1:10 or 1:100 dilution) for 1 h at
37°C. At the end of the incubation, plates were washed and incubated
with a monoclonal anti-pPG antibody-biotin conjugate (16)
(1:1,000 dilution) at 37°C for 30 min, and then they were washed and
incubated with avidin-horseradish peroxidase conjugate (1:1,000
dilution) at 37°C for 30 min. After washing, plates were developed
with o-phenylenediamine substrate (Sigma) and the optical densities at 492 nM were measured with a microplate reader. Standards and samples were run in triplicate.
For Western blot analysis, after electrophoresis, proteins were
electroblotted to an Immobilon-P membrane in 0.7% acetic acid
and the
blots were probed with a 1:1,000 dilution of the monoclonal
anti-PG3
antibody and a 1:1,000 dilution of rabbit anti-mouse
immunoglobulin
G-alkaline phosphatase conjugate, then developed
in a
5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium
solution.
Recombinant PG3 (
16) was used as a positive control.
Elastase activity and elastase inhibitory capacity assays.
The elastase activity assay and elastase inhibitory capacity assay were
performed by using Alphasin elastase diffusion plates (Elastin Products
Co., Owensville, Mo.) as described by the manufacturer. An Alphasin
elastase diffusion plate is a square plastic petri dish containing 10 ml of buffered agar gel that has been uniformly impregnated with
elastin-fluorescein with a particle size smaller than 37 µm. The gel
contains 25 wells, each 4 mm in diameter. Elastase and purified 
1
protease inhibitor (1-PI) were dissolved in 0.15 M NaCl. An aliquot
(10 µl) of elastase (400 ng), alone or mixed with a series of
concentrations of either 1-PI or serum, or with neutrophil
secretion, was added to each well. Samples were run in duplicate.
Plates were incubated at 37°C for 6 h, and the diameter of the
clear zone (resulting from the solubilization of elastin particles by
elastase) was measured. The elastase inhibitory capacities of
neutrophil secretion and serum were determined by using 1-PI as the
standard.
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RESULTS |
Elastase is required for the generation of neutrophil secretions
bactericidal to E. coli.
Initially, we determined the
contribution of elastase-cleaved polypeptides to bactericidal
activity released from freshly isolated porcine neutrophils.
The neutrophils were incubated at 108/ml in PBS
containing 1 mM Ca2+ and 1 mM Mg2+ and were
stimulated for 30 min with PMA (100 ng/ml) in the presence or the
absence of the specific inhibitor for neutrophil elastase, CMK. The
bactericidal activity of the cell-free secretions was assayed after
overnight storage at 4°C.
Secretions of unstimulated neutrophils were not bactericidal to either
L. monocytogenes or
E. coli (Fig.
1), but upon stimulation
by PMA,
porcine neutrophils secreted antibacterial substances
that were
bactericidal to both
L. monocytogenes and
E. coli. The
antibacterial activity of neutrophil secretions against
both bacteria
was completely blocked if CMK, the specific
elastase inhibitor,
was added before neutrophils were stimulated by
PMA, but not if
CMK was added after incubation with PMA. We had shown
previously
(
16) that CMK had no detectable effect on
bacteria and no effect
on antibacterial activity when added to
cell-free polymorphonuclear
leukocyte (PMN) secretions. The inhibition
of bactericidal activity
by CMK is consistent with the proposed role of
elastase as an
activator of secreted cathelicidins for extracellular
killing
of bacteria.
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FIG. 1.
Bactericidal activity of neutrophil secretions in a
radial diffusion assay. Five microliters of neutrophil secretion
(equivalent to that from 5 × 105 neutrophils) was
added into each well. Secretions were obtained from unstimulated
neutrophils (N), neutrophils stimulated with PMA (100 ng/ml) for 30 min
in the absence of CMK, with CMK added to the secretion after
neutrophils were removed [(N+P)+C], or neutrophils stimulated with
PMA in the presence of CMK (N+C+P). Zones of clearance were measured
with a calibrated microscope. The microbicidal activity was expressed
in arbitrary units (0.1 mm = 1 U) by subtracting the diameter of
the well (3 mm) from the diameter of the clear zone. Open bars,
E. coli; solid bars, L. monocytogenes. Data are
means ± standard deviations from assays performed twice in
duplicate.
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Antibacterial activity of neutrophil secretions persists in the
presence of normal salt medium and 1 to 2% serum.
While the
effect of porcine serum on the antibacterial activity of neutrophil
secretions is potentially very complex (10, 11, 15, 17), the
microbicidal activity of PG1 has been reported to be resistant to serum
(21, 27). Indeed, we confirmed that the bactericidal
activity of PG3 against both L. monocytogenes and E. coli was also unchanged or even increased in the presence of 1 to
2% porcine serum and normal salt medium compared to that under
low-salt, serum-free conditions (data not shown).
An important effect of serum is mediated by its neutrophil elastase
inhibitors, e.g.,
1-PI and
2-macroglobulin, which could
impair
the proteolytic processing of protegrins and other elastase-activated
polypeptides and could decrease the antibacterial activity of
neutrophil secretions. To assess the effect of serum on the
microbicidal
activity of neutrophil secretions, serum was added either
before
the PMA-induced secretion of granule contents or, as a control,
after the degranulation was completed and the neutrophils were
removed.
Regardless of when the serum was added, it did not inhibit,
and in some
cases it enhanced, the microbicidal activity of neutrophil
secretions
against both
E. coli and
L. monocytogenes (Fig.
2).
Porcine serum by itself was inactive
against both bacteria under
these assay conditions. The protein
composition of neutrophil
secretions was assessed by AU-PAGE (Fig.
3). In the presence of
1 to 2% serum,
the proteolytic generation of protegrins from pPGs
was not inhibited.
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FIG. 2.
Influence of serum on the antibacterial activity of
neutrophil secretions in a radial diffusion assay. N+S+P, neutrophils
were stimulated by PMA (100 ng/ml) in the presence or absence of serum;
S, serum only, diluted in PBS; (N+P)+S, serum was added into neutrophil
secretions after neutrophils were removed. Symbols:  , E. coli;  , L. monocytogenes. Data are means ± standard deviations from assays performed twice in duplicate.
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FIG. 3.
AU-PAGE of neutrophil secretions stimulated by PMA in
the presence or absence of serum. Proteins and peptides from neutrophil
secretions or serum solutions were subjected to AU-PAGE and stained
with Coomassie blue after electrophoresis. Recombinant pPG3 (upper
band) and synthetic PG3 (lower band) (0.5 µg of each) were used as
standards (leftmost lane). In neutrophil secretions, the bands
comigrating with the PG3 standard contain PG2 and PG3, and the slightly
faster migrating band corresponds to PG1. Neutrophil secretions were
induced by PMA in the presence or absence of serum. For each lane, 100 µl of neutrophil secretions (equivalent to that from 107
cells) or 100 µl of serum solution (1 or 2%) was extracted with 10 µl of StrataClean resin (Stratagene) and the resin beads were loaded
into each well for electrophoresis.
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We next asked whether the inability of serum to inhibit the processing
of protegrins by neutrophil elastase could be due to
the lack of
elastase inhibitory activity in porcine serum. The
elastase inhibitory
capacities of serum and of neutrophil secretions
were assayed on
elastin plates relative to human
1-PI as a standard
inhibitor of
elastase. As shown in Fig.
4, the
elastase inhibitory
capacity in 10% normal fresh porcine serum was
equivalent to that
of 70 µg of
1-PI/ml, a finding similar to
reported data (
23).
However, exposure of 1 to 20% serum to
neutrophils activated by
PMA completely ablated the elastase inhibitory
capacity. The loss
of elastase inhibitory capacity is likely due to the
inactivation
of
1-PI and other elastase inhibitors by reactive
oxygen intermediates
generated during the respiratory burst of
neutrophils (
2).
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FIG. 4.
The elastase inhibitory capacity (EIC) of porcine serum.
Human neutrophil elastase (400 ng) in 10 µl of saline (0.15 M NaCl)
or saline-serum mixture (serum), or a mixture of elastase with serum
exposed to activated porcine neutrophils (conditioned serum), was
assayed on an elastin-agar plate. Mixtures of human neutrophil elastase
(400 ng) with varying amounts of purified 1-PI were used as
standards for the inhibition of elastase activity. Data are means from
two experiments.
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During phagocytosis, protegrins are generated from pPGs by
elastase.
To study the role of protegrins and other
elastase-activated peptides in secretions generated during
phagocytosis, we first incubated porcine neutrophils with zymosan that
had been opsonized with porcine serum. Phagocytosis was verified by
light microscopy. However, mature protegrins were not detected in the
neutrophil secretions by AU-PAGE analysis (data not shown), and to our
surprise, no bactericidal activity was found in the zymosan-stimulated
neutrophil secretions by the radial diffusion assay and CFU assay (data
not shown). In a previous study, we found that the extracellular
processing of pPGs by PMA was mediated by neutrophil elastase, since it
could be inhibited extracellularly by the specific inhibitor CMK
(16). To determine whether elastase cleaved pPGs during
neutrophil phagocytosis of zymosan, we used the cell-permeant serine
protease inhibitor DFP to inhibit neutrophil elastase (and other serine
proteases) during phagocytosis (1), or we used CMK as an
extracellular elastase-specific inhibitor. Light microscopy revealed
that DFP- or CMK-treated PMNs phagocytized the same number of zymosan
particles as untreated PMNs (data not shown), in agreement with
observations reported by others (13). To detect the
processed and unprocessed protegrin forms, we performed both Western
blot analysis and an ELISA using anti-pPG and antiprotegrin antibodies.
In Western blot analysis, only pPGs, not protegrins, were detected in
neutrophil secretions when DFP or CMK was added with zymosan, but
without DFP and CMK, neither pPGs nor protegrins were detected (data
not shown). By an ELISA designed to detect pPGs only, the amounts of
pPGs released from neutrophils during phagocytosis of zymosan were
22.29 ± 2.28 µg/108 PMNs/ml and 23.90 ± 0.70 µg/108 PMNs/ml in the presence of DFP and CMK
respectively, which were similar to those resulting from PMA
stimulation in the presence of CMK (21.57 ± 3.10 µg/108 PMNs/ml). In the absence of CMK and DFP, only
small amounts of pPGs were detectable in secretions generated by either
PMA- or zymosan-treated neutrophils (1.35 ± 0.13 µg/108 PMNs/ml for PMA versus 1.55 ± 0.07 µg/108 PMNs/ml for zymosan). We concluded that elastase
did process secreted pPGs during phagocytosis of zymosan.
We hypothesized that our inability to detect processed protegrins was
due to their high affinity for zymosan, leading to the
adsorption of
protegrins from the neutrophil secretions. To test
this hypothesis, we
added zymosan either to neutrophil secretions
that had been generated
by PMA-treated neutrophils or to a solution
of synthetic PG3. After 30 min of incubation at 37°C, zymosan
particles were removed by brief
centrifugation, and the supernatants
were tested for antibacterial
activity against both
E. coli and
L. monocytogenes. While solutions not exposed to zymosan were
microbicidal to both bacteria, no antibacterial activity was found
in
the zymosan-extracted neutrophil secretions or PG3 solutions
(data not
shown). The interaction of protegrins with zymosan was
so strong that
protegrins could not be detected even when the
zymosan-protegrin
complex was subjected to AU-PAGE and sodium
dodecyl sulfate-PAGE under
reducing conditions (data not shown).
To overcome the difficulty caused by protegrins binding to zymosan
particles, we used latex beads as an alternative particulate
stimulus.
First, we assayed the binding of PG3 to latex beads
and found that PG3
bound to the latex less avidly than to zymosan
and that bound PG3 was
released when the latex-PG3 complex was
subjected to AU-PAGE (data not
shown). After phagocytosis of latex,
neutrophils were sedimented by
low-speed centrifugation (at 200
×
g for 6 min),
leaving neutrophil secretions and the uningested
latex beads in the
supernatant. Latex beads were then removed
from the neutrophil
secretions by high-speed centrifugation at
13,000 ×
g
for 1 min, leaving latex-free neutrophil secretions.
The fractions were
analyzed by AU-PAGE. As shown in Fig.
5,
protegrins
were detected in all the fractions from neutrophils that
phagocytized
latex beads but received the cell-permeant serine protease
inhibitor
DFP only at the completion of phagocytosis, but no protegrins
were detected if DFP was added before latex beads were incubated
with
neutrophils. No protegrins were detected in unstimulated
neutrophils
where latex beads were added at the end of the incubation.
The
activation of protegrins during neutrophil phagocytosis of
latex beads
was further confirmed by Western blot analysis using
a monoclonal
antiprotegrin antibody (Fig.
6). Porcine
neutrophil
granules, when isolated without protease inhibitors, contain
both
mature protegrins and their precursors. However, only mature
protegrins
were detected in the neutrophil secretions induced by
phagocytosis
of latex beads. The presence of mature protegrins in the
cell
lysates of neutrophils that phagocytized latex beads suggested
that either protegrins were activated from their precursors inside
or
on the surfaces of neutrophils or they were adsorbed secondarily
from
the medium (Fig.
5).
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FIG. 5.
AU-PAGE of neutrophil secretions stimulated by latex
beads. Gels were stained with Coomassie blue. Each lane contains either
107 cell equivalents of either neutrophil secretions,
extracellular latex beads after phagocytosis, or neutrophil lysates.
The symbols + and indicate which materials were added into
each tube before the phagocytosis started. At the end of the
incubation, DFP and/or latex beads were added into tubes that did not
have them during phagocytosis. The PG standard is 0.5 µg of PG3.
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FIG. 6.
Western blot analysis of neutrophil secretions
stimulated by latex beads. After AU-PAGE, proteins were blotted to an
Immobilon-P membrane, and the membrane was probed with a monoclonal
anti-PG3 antibody and rabbit anti-mouse immunoglobulin G-alkaline
phosphatase conjugate, then developed in
5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium solution.
PG3, 0.5 µg of synthetic PG3; G, porcine neutrophil granule lysate
(5 × 106 cell equivalents); S, neutrophil secretions
generated during phagocytosis of latex beads in the absence of DFP
(107 cell equivalents).
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The antibacterial activity of neutrophil secretions stimulated by latex
beads was evaluated by a CFU assay and a gel-overlay
assay. As shown in
Fig.
7, neutrophils secreted
antibacterial
substances that were active against
L. monocytogenes, and this
activity was inhibited when DFP was added
before the neutrophils
phagocytized latex beads. In the gel overlay
assay, when either
E. coli or
L. monocytogenes
was used to detect antimicrobial activity,
the results were very
similar (Fig.
8). Activity corresponding
to protegrins was seen only in secretions generated by neutrophils
that
had not been pretreated with DFP. Additional antibacterial
bands were
detected in the neutrophil secretions, and these were
slower migrating
than protegrins. Because the activity of these
substances was also
blocked by pretreatment with DFP, we suspect
that these bands represent
other elastase-activated cathelicidins,
such as PR-39 (
19),
prophenins (
5), and PMAPs (
24).
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FIG. 7.
Bactericidal activity of latex-stimulated neutrophil
secretions in a CFU assay. Shown are the results of a CFU assay with
L. monocytogenes. T0, initial bacterial inoculum; T30,
bacteria in PBS after 30 min at 37°C. Bacteria were incubated as
follows: with neutrophil secretions stimulated with latex beads for 30 min and then treated with DFP (L); with neutrophil secretions
stimulated with latex beads for 30 min in the presence of DFP (D/L);
with neutrophil secretions elicited with DFP, incubated for 30 min, and
then treated with latex beads at the end of incubation (D); or with
neutrophil secretions treated with DFP and latex beads at the end of 30 min of incubation (O).
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FIG. 8.
Bactericidal activity of latex-stimulated neutrophil
secretions in a gel-overlay bactericidal assay. Shown are results of
two gel-overlay assays with E. coli and L. monocytogenes. Neutrophil secretions were prepared as described
for Fig. 5. Ten million cell equivalents of neutrophil secretion was
used in each lane of the overlay assays. PG, 0.5 µg of synthetic PG3,
used as positive control for the activity and identification of
protegrins. Antibacterial activity was indicated by a clear zone (no
bacterial growth).
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|
Protegrins contribute to the bactericidal activity of abscess
fluid.
Abscesses contain accumulations of activated neutrophils
and serum proteins. To determine whether cell-free porcine abscess fluid is microbicidal, we collected fresh abscess fluid from two pigs,
one with an abdominal wall abscess (A1) and one with a dermal abscess
(A2), rapidly removed the particulate fraction by centrifugation, and
assayed the microbicidal activity of the supernatant by radial diffusion assay. One abscess fluid sample (A2) displayed potent activity (100 to 120 radial diffusion units) against both E. coli and L. monocytogenes; however, the activity of the
other sample was very weak (10 to 15 radial diffusion units). To detect
microbicidal protegrins, we then analyzed the supernatant using
AU-PAGE, a gel-overlay bactericidal assay, and Western blot analysis
(Fig. 9). The protein profiles of abscess
fluids on AU-PAGE indicated that protegrins were present in both
samples. The activity and the immunoreactivity of protegrins in the
abscess fluids were confirmed by gel-overlay bactericidal assay and
Western blot analysis using a monoclonal antiprotegrin antibody (Fig.
9). Based on the intensity of the Western blot, we estimated that the
concentrations of mature protegrins in abscess fluids were 15 to 150 µg/ml, well within the range of concentrations known to be
bactericidal to E. coli and L. monocytogenes
(9). The Western blot also indicated that most of the
protegrins in the less-active abscess fluid were strongly associated
with substances that prevented the normal migration of protegrins in
AU-PAGE. Additional studies will be necessary to identify these
substances and to determine whether they originate in the microbes or
the host.
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FIG. 9.
Microbicidal activity of porcine abscess fluids. Porcine
abdominal-wall abscess fluid (A1) (20 µl), porcine skin abscess fluid
(A2) (20 µl), and synthetic PG3 (0.03, 0.1, and 0.3 µg) were
subjected to AU-PAGE. (Left) The gel was stained with Coomassie blue.
(Center) Gel-overlay assay with E. coli. Antibacterial
activity was indicated by a clear zone (no bacterial growth). (Right)
Western blot analysis was performed as for Fig. 6.
|
|
| |
DISCUSSION |
The role of an individual peptide in the neutrophil antimicrobial
activity has been difficult to determine because several families of
antimicrobial proteins and peptides typically coexist in the
neutrophils of each mammalian species. Thus, both defensins and
cathelicidins have been identified in the neutrophils of humans, rabbits, and cattle. We selected porcine neutrophils as a simple model
for the study of cathelicidins, since the porcine neutrophils lack
defensins (9). Evidence supporting the hypothesis that neutrophil cathelicidins are important host defense factors is emerging. We previously showed that protegrins were stored as inactive
proforms in porcine neutrophil granules and could be activated
extracellularly by elastase. Stimulation of neutrophil secretions with
PMA rendered them bactericidal to L. monocytogenes in
low-salt medium (16). In other studies, rabbit cathelicidins referred to as p15's potently synergized with BPI to inhibit the growth of E. coli in rabbit peritoneal exudate
(25). These two rabbit cathelicidins, p15a and p15b, have
less-anionic cathelin domains than other cathelicidins, lack the
characteristic elastase cleavage sites, and do not require
elastase-mediated activation. In the present study, we demonstrated
that the antibacterial activity of porcine neutrophil secretions
generated during neutrophil phagocytosis of latex beads was dependent
in large part on elastase-activated polypeptides, including protegrins.
The elastase-dependent activity was detected against both E. coli and L. monocytogenes under normal-salt conditions.
Protegrins, active and abundant porcine cathelicidins, were found at
bactericidal concentrations in neutrophil secretions and in abscess
fluid. Further studies will be necessary to identify the other
elastase-activated polypeptides, to ascertain whether they are also
cathelicidins, and to assess their contribution to the antibacterial
activity of porcine inflammatory fluid.
In vivo, neutrophils are commonly exposed to varying concentrations of
serum, which contains elastase inhibitors. The extracellular processing
of pPGs and other procathelicidins to active forms must depend on the
release of elastase in functional excess of its inhibitors. Despite the
strong elastase inhibitory activity in fresh porcine serum, the
maturation of protegrins was observed even when the serum was added
before neutrophil activation. We found that the elastase inhibitory
activity of fresh porcine serum was totally abolished during
neutrophil activation, probably by respiratory-burst products
(2). Thus, in the neighborhood of phagocytizing neutrophils,
elastase may exceed its inhibitors and activate cathelicidins. The
unimpaired processing of protegrins could also be explained by the
adsorption of elastase to the neutrophil surface, which can protect
elastase from its inhibitors (14).
Protegrins, and other elastase-activated polypeptides, were the
predominant antibacterial factors in porcine neutrophil secretions generated during phagocytosis. Although species differences in the
antimicrobial arsenal of neutrophils preclude direct extrapolation to
humans, the potential host defense role of elastase as an activating enzyme for the precursors of microbicidal peptides must be taken into
account when therapeutic inhibitors of neutrophil elastase are
evaluated for clinical use as anti-inflammatory agents (25).
| |
ACKNOWLEDGMENTS |
We thank Shawn McGill, Fernando Vinuela, Jr., and John Robert for
their excellent technical support and Edith Martin Porter for helpful
discussions.
This work was supported by grant HL46809 from the National Institutes
of Health and a postdoctoral fellowship from the Cystic Fibrosis
Foundation to J. Shi.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medicine, CHS 37-055, 10833 Le Conte Ave., UCLA School of Medicine, Los Angeles, CA 90095. Phone: (310) 825-6112. Fax: (310) 206-8766. E-mail: tganz{at}ucla.edu.
Editor: R. N. Moore
| |
REFERENCES |
| 1.
|
Amrein, P. C., and T. P. Stossel.
1980.
Prevention of degradation of human polymorphonuclear leukocyte proteins by diisopropylfluorophosphate.
Blood
56:442-447[Abstract/Free Full Text].
|
| 2.
|
Carp, H., and A. Janoff.
1980.
Potential mediator of inflammation. Phagocyte-derived oxidants suppress the elastase-inhibitory capacity of alpha 1-proteinase inhibitor in vitro.
J. Clin. Investig.
66:987-995.
|
| 3.
|
Ganz, T., and R. I. Lehrer.
1997.
Antimicrobial peptides of leukocytes.
Curr. Opin. Hematol.
4:53-58[Medline].
|
| 4.
|
Ganz, T., and J. Weiss.
1997.
Antimicrobial peptides of phagocytes and epithelia.
Semin. Hematol.
34:343-354[Medline].
|
| 5.
|
Harwig, S. S.,
V. N. Kokryakov,
K. M. Swiderek,
G. M. Aleshina,
C. Zhao, and R. I. Lehrer.
1995.
Prophenin-1, an exceptionally proline-rich antimicrobial peptide from porcine leukocytes.
FEBS Lett.
362:65-69[Medline].
|
| 6.
|
Henson, P. M.
1971.
The immunologic release of constituents from neutrophil leukocytes. II. Mechanisms of release during phagocytosis, and adherence to nonphagocytosable surfaces.
J. Immunol.
107:1547-1557[Abstract/Free Full Text].
|
| 7.
|
Jandl, R. C.,
J. Andre-Schwartz,
L. Borges-DuBois,
R. S. Kipnes,
B. J. McMurrich, and B. M. Babior.
1978.
Termination of the respiratory burst in human neutrophils.
J. Clin. Investig.
61:1176-1185.
|
| 8.
|
Kokryakov, V. N.,
S. S. Harwig,
E. A. Panyutich,
A. A. Shevchenko,
G. M. Aleshina,
O. V. Shamova,
H. A. Korneva, and R. I. Lehrer.
1993.
Protegrins: leukocyte antimicrobial peptides that combine features of corticostatic defensins and tachyplesins.
FEBS Lett.
327:231-236[Medline].
|
| 9.
|
Lehrer, R. I., and T. Ganz.
1996.
Endogenous vertebrate antibiotics. Defensins, protegrins, and other cysteine-rich antimicrobial peptides.
Ann. N. Y. Acad. Sci.
797:228-239[Medline].
|
| 10.
|
Lehrer, R. I.,
D. Szklarek,
A. Barton,
T. Ganz,
K. J. Hamann, and G. J. Gleich.
1989.
Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein.
J. Immunol.
142:4428-4434[Abstract].
|
| 11.
|
Lehrer, R. I.,
D. Szklarek,
T. Ganz, and M. E. Selsted.
1985.
Correlation of binding of rabbit granulocyte peptides to Candida albicans with candidacidal activity.
Infect. Immun.
49:207-211[Abstract/Free Full Text].
|
| 12.
|
Moscinski, L. C., and B. Hill.
1995.
Molecular cloning of a novel myeloid granule protein.
J. Cell. Biochem.
59:431-442[Medline].
|
| 13.
|
Musson, R. A., and E. L. Becker.
1977.
The role of an activatable esterase in immune-dependent phagocytosis by human neutrophils.
J. Immunol.
118:1354-1365[Abstract/Free Full Text].
|
| 14.
|
Owen, C. A.,
M. A. Campbell,
P. L. Sannes,
S. S. Boukedes, and E. J. Campbell.
1995.
Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases.
J. Cell Biol.
131:775-789[Abstract/Free Full Text].
|
| 15.
|
Panyutich, A., and T. Ganz.
1991.
Activated alpha 2-macroglobulin is a principal defensin-binding protein.
Am. J. Respir. Cell Mol. Biol.
5:101-106.
|
| 16.
|
Panyutich, A.,
J. Shi,
P. L. Boutz,
C. Zhao, and T. Ganz.
1997.
Porcine polymorphonuclear leukocytes generate extracellular microbicidal activity by elastase-mediated activation of secreted proprotegrins.
Infect. Immun.
65:978-985[Abstract].
|
| 17.
|
Panyutich, A. V.,
P. S. Hiemstra,
S. Van Wetering, and T. Ganz.
1995.
Human neutrophil defensin and serpins form complexes and inactivate each other.
Am. J. Respir. Cell Mol. Biol.
12:351-357[Abstract].
|
| 18.
|
Shi, J.,
R. D. Goodband,
M. M. Chengappa,
J. L. Nelssen,
M. D. Tokach,
D. S. McVey, and F. Blecha.
1994.
Influence of interleukin-1 on neutrophil function and resistance to Streptococcus suis in neonatal pigs.
J. Leukocyte Biol.
56:88-94[Abstract].
|
| 19.
|
Shi, J.,
C. R. Ross,
M. M. Chengappa, and F. Blecha.
1994.
Identification of a proline-arginine-rich antibacterial peptide from neutrophils that is analogous to PR-39, an antibacterial peptide from the small intestine.
J. Leukocyte Biol.
56:807-811[Abstract].
|
| 20.
|
Sorensen, O.,
K. Arnljots,
J. B. Cowland,
D. F. Bainton, and N. Borregaard.
1997.
The human antibacterial cathelicidin, hCAP-18, is synthesized in myelocytes and metamyelocytes and localized to specific granules in neutrophils.
Blood
90:2796-2803[Abstract/Free Full Text].
|
| 21.
|
Steinberg, D. A., and R. I. Lehrer.
1997.
Designer assays for antimicrobial peptides, p. 169-187.
In
W. M. Shafer (ed.), Antimicrobial peptide protocols. Humana Press, Inc., Totowa, N.J.
|
| 22.
|
Storici, P.,
A. Tossi,
B. Lenarcic, and D. Romeo.
1996.
Purification and structural characterization of bovine cathelicidins, precursors of antimicrobial peptides.
Eur. J. Biochem.
238:769-776[Medline].
|
| 23.
|
Stratil, A.,
D. Cizova-Schroffelova,
E. Gabrisova,
M. Pavlik,
W. Coppieters,
L. Peelman,
A. Van de Weghe, and Y. Bouquet.
1995.
Pig plasma alpha-protease inhibitors PI2, PI3 and PI4 are members of the antichymotrypsin family.
Comp. Biochem. Physiol. B
111:53-60[Medline].
|
| 24.
|
Tossi, A.,
M. Scocchi,
M. Zanetti,
P. Storici, and R. Gennaro.
1995.
PMAP-37, a novel antibacterial peptide from pig myeloid cells. cDNA cloning, chemical synthesis and activity.
Eur. J. Biochem.
228:941-946[Medline].
|
| 25.
|
Vender, R. L.
1996.
Therapeutic potential of neutrophil-elastase inhibition in pulmonary disease.
J. Investig. Med.
44:531-539[Medline].
|
| 26.
|
Weinrauch, Y.,
A. Foreman,
C. Shu,
K. Zarember,
O. Levy,
P. Elsbach, and J. Weiss.
1995.
Extracellular accumulation of potently microbicidal bactericidal/permeability-increasing protein and p15s in an evolving sterile rabbit peritoneal inflammatory exudate.
J. Clin. Investig.
95:1916-1924.
|
| 27.
|
Yasin, B.,
S. S. Harwig,
R. I. Lehrer, and E. A. Wagar.
1996.
Susceptibility of Chlamydia trachomatis to protegrins and defensins.
Infect. Immun.
64:709-713[Abstract].
|
| 28.
|
Zanetti, M.,
R. Gennaro, and D. Romeo.
1995.
Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain.
FEBS Lett.
374:1-5[Medline].
|
| 29.
|
Zanetti, M.,
L. Litteri,
G. Griffiths,
R. Gennaro, and D. Romeo.
1991.
Stimulus-induced maturation of probactenecins, precursors of neutrophil antimicrobial polypeptides.
J. Immunol.
146:4295-4300[Abstract].
|
Infect Immun, August 1998, p. 3611-3617, Vol. 66, No. 8
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
