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Divisions of
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Periodontics and Endodontics and
Microbiology and Immunology, Tohoku University Graduate School of Dentistry, Sendai, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, indicating that HLE
inhibited a CD14-dependent cell activation. These findings suggested
that activated PMNs have a potential negative feedback mechanism for
HGF function at the inflammatory site, particularly in periodontal
tissues. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Connective tissue is a major source of fibroblasts, which are a common cell type to most tissues and organs, and are implicated in the pathological production of extracellular matrix and scar tissue in fibrotic inflammatory diseases. Fibroblasts not only function in the support of frameworks by synthesis of extracellular matrix such as collagens, fibronectin, and laminin, but also actively participate in inflammatory and immune responses by producing cytokines such as IL-1, IL-6, and chemokines, in particular IL-8 (7). Human gingival fibroblasts (HGF) produce IL-8 upon stimulation with LPS in a membrane CD14-dependent manner (8).
CD14, a 55-kDa GPI-anchored protein (9), is mainly expressed on monocytes and macrophages, and heterogeneously expressed on HGF, as reported previously (8). CD14 functions not only as a major receptor for LPS from Gram-negative bacteria (9), but also acts as a pattern recognition receptor for many bacterial components, such as lipoteichoic acids from Gram-positive bacteria, lipoarabinomannan from mycobacteria, soluble peptidoglycan from Staphylococcus aureus, and lipoproteins from Treponema pallidum and Borrelia burgdorferi (10, 11). Cell activation triggered by the bacterial components via CD14 is thought to be the first line of defense against invasive bacteria.
Activated PMNs express many molecules on the cell surface (12) and release soluble factors such as cytokines (13) and proteases (1), and interactions between fibroblasts and infiltrating PMNs have been observed at inflammatory sites (14). Although these interactions have been widely investigated with regard to the mechanism of adhesion of PMNs to fibroblasts (15, 16, 17), little is known about regulation of fibroblast function influenced by activated PMNs. To help clarify this, we used a coculture system with PMNs and HGF, and examined how activated PMNs could influence the function of HGF with regard to cytokine production. We found that activated PMNs cleaved CD14 on HGF by direct proteolysis, and this proteolysis was mediated particularly by human leukocyte elastase (HLE, E.C. 3.4.21.11) released from activated PMNs, which resulted in down-regulation of IL-8 production by HGF triggered by LPS in a CD14-dependent manner.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LPS of Escherichia coli O55:B5, HYBRI-MAX (serum-free
and protein-free medium), 1-antitrypsin (1-AT), PMSF,
1,10-phenanthroline, FMLP, and PMA were purchased from Sigma (St.
Louis, MO). Bacillus cereus phosphatidylinositol-specific
phospholipase C (PI-PLC), pepstatin (an asparatic protease inhibitor),
and E-64
(N-[N-(L-3-trans-carboxirane-2-carbonyl)-L-leucyl]agmatine,
a cysteine protease inhibitor) were purchased from Boehringer Mannheim
(Indianapolis, IN). FCS was purchased from Flow Laboratories (McLean,
VA). HLE, human neutrophil cathepsin G, and
N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone
(HLE/CMK) were purchased from Calbiochem-Novabiochem (La Jolla, CA).
Human leukocyte proteinase 3 (PR3) was purchased from HyTest (Turku,
Finland). -MEM and 0.25% trypsin/1 mM EDTA were from Life
Technologies (Rockville, MD). Human rIL-1 and human rTNF- were
obtained from Dainippon Pharmaceutical (Osaka, Japan). Anti-HLA-ABC
FITC (G46-2.6, mouse IgG1), anti-CD59 FITC (p282, mouse IgG2a), and
anti-CD26 mAb (M-A261, mouse IgG1) were from PharMingen (San Diego,
CA). Anti-CD14 mAb (MEM-18, mouse IgG1) was from Monosan (Uden, The
Netherlands). Anti-CD14 mAb (MY4, mouse IgG2b), anti-CD157 mAb
(RF3, mouse IgG1), and all isotype control mAbs were obtained from
Immunotech (Marseille, France). All other reagents were obtained from
Sigma, unless otherwise indicated.
Isolation of human PMNs
PMNs from heparinized (10 U/ml) peripheral venous blood were isolated by density-gradient centrifugation on Mono-Poly resolving medium (ICN Biomedical, Costa Mesa, CA) at 300 x g for 30 min at room temperature (18). The fraction containing PMNs was harvested and washed twice with PBS at 4°C. The viability of these cells was greater than 98%, as judged by trypan blue dye exclusion. The purity of PMNs was above 95% morphologically.
Human gingival fibroblast
HGF were prepared from the explants of normal gingiva from 8- to
25-year-old patients with informed consent, as reported previously
(19). Explants were cut into pieces and cultured in
100-mm-diameter tissue culture dishes (Falcon; Becton Dickinson
Labware, Lincoln Park, NJ) in -MEM supplemented with 10% FCS with a
medium change every 3 days for 10–15 days until confluent cell
monolayers were formed. The cells were detached with 0.25% trypsin/1
mM EDTA, washed with PBS, and subcultured in plastic flasks (Corning
Coster, Acton, MA). After three to four subcultures by trypsinization,
homogeneous, slim, spindle-shaped cells grown in characteristic swirls
were obtained. The cells were used as confluent monolayers at
subculture levels 5 through 15.
Flow cytometry
HGF were collected by trypsinization, washed with PBS (pH 7.2), and used for staining. For HLA-ABC and CD59 staining, 105 HGF were stained with FITC-conjugated mAbs or FITC-conjugated isotype-matched mouse IgG at 4°C for 30 min. For CD14, CD157, and CD26 staining, cells were stained with each mAb or each isotype-matched control IgG at 4°C for 30 min, followed by incubation with FITC-conjugated goat anti-mouse IgG (BioSource International, Camarillo, CA) at 4°C for an additional 30 min. Staining was analyzed on a FACScan (Becton Dickinson, Mountain View, CA). For staining fixed HGF, monolayers of HGF in 24-well multiplates were treated with 1% (w/v) paraformaldehyde for 20 min at room temperature. After washing three times with PBS, HGF were treated with HLE, as described below, and harvested by trypsinization and then stained with anti-CD14 mAb. In the coculture experiment of HGF with PMNs, the HGF population was gated on the basis of forward and right-angle scatters. Data were collected for 5000 events, which were stored in list mode and then analyzed with Lysis II software (Becton Dickinson). The arithmetic mean was used in the computation of the mean fluorescence intensity (MFI).
Coculture of HGF monolayer with PMNs
Confluent monolayers of HGF (approximately
105 cells) in collagen I (rat tail tendon)-coated
24-well multiplates (Becton Dickinson Labware) were cocultured with the
indicated number of purified PMNs in 500 µl of FCS-free -MEM with
or without 100 ng/ml PMA for 60 min at 37°C. Cells were collected by
trypsinization, and expression of CD14 by HGF was analyzed by flow
cytometry. To harvest the cell-free supernatants from PMNs, the
indicated number of purified PMNs in a microtube containing 500 µl of
FCS-free -MEM were stimulated with 100 ng/ml PMA for 60 min at
37°C or primed with 100 ng/ml TNF- for 10 min, followed by
stimulation with 1 µM FMLP for 60 min at 37°C. After incubation of
PMNs, supernatants were harvested by centrifugation, and kept at
-20°C until use. For fixing PMNs, PMNs were stimulated with PMA
similar to cell-free supernatant and fixed for 3 min at 4°C in PBS
containing 3% (w/v) paraformaldehyde and 0.25% (v/v) glutaraldehyde
(pH 7.4). After washing twice in HBSS, the indicated number of fixed
PMNs were resuspended with 500 µl of FCS-free -MEM. For protease
inhibitor treatment, the indicated concentration of each inhibitor was
preincubated with cell-free supernatant (500 µl) from 3 x
106 PMNs or fixed PMNs (3 x
106/500 µl) for 10 min at 37°C before
addition onto monolayer cells. Percent inhibition for the supernatant
and the fixed cells was calculated as ((MFI in the presence of
supernatant and inhibitors) - (MFI in the presence of
supernatant))/((MFI in the absence of supernatant) - (MFI in the
presence of supernatant)) x 100 (%) and [(MFI in the presence
of fixed PMNs and inhibitors) - (MFI in the presence of fixed
PMNs)]/[(MFI in the absence of fixed PMNs) - (MFI in the
presence of fixed PMNs)] x 100 (%), respectively.
HLE treatment
Monolayers of HGF in collagen I-coated 24-well multiplates (the
well contained 300 µl of serum-free media, HYBRI-MAX) were treated
with the indicated concentration of HLE at 37°C for the indicated
times. For HLE inhibitor (1-AT and human serum) treatments, 20
µg/ml HLE was preincubated with 100 µg/ml 1-AT or 10% (v/v)
human serum for 15 min at 37°C before addition onto monolayer
cells.
Reexpression of CD14 following proteolytic cleavage
Confluent monolayers of HGF in wells of 24-well collagen I-coated plates were treated with or without 20 µg/ml HLE in HYBRI-MAX for 30 min at 37°C. After gently washing monolayers with prewarmed HYBRI-MAX, 1 ml of prewarmed HYBRI-MAX with 0.5% FCS was added to the wells and incubated at 37°C. At the indicated times, cells were analyzed for the expression of CD14 by flow cytometry.
Preparation of cell membranes and treatment with enzymes
HGF were collected by trypsinization, and cells were suspended in hypotonic buffer (10 mM Tris-HCl, pH 7.4, and 1 mM MgCl2) and incubated on ice for 30 min. Cells were then homogenized in a Dounce homogenizer by 15 strokes, and sucrose was added to a final concentration of 0.25 M. The homogenate was centrifuged at 500 x g for 5 min twice to remove nuclei. Supernatants were centrifuged at 15,000 x g for 30 min, and membrane pellets were suspended in PBS and stored at -20°C until use.
Western blotting
Cell membranes from confluent HGF grown in 25-cm2 flasks were suspended in 100 µl of HBSS containing 20 µg/ml HLE or 5 U/ml PI-PLC and treated for 60 min at 37°C. Membrane pellets and supernatants were separated by centrifugation at 15,000 x g for 30 min at 4°C, and supernatants were dried by vacuum centrifugation. Membrane pellets and dried supernatants were solubilized with 20 µl of Laemmli sample buffer (10% glycerol, 1% SDS, 0.0025% bromphenol blue, and 50 mM Tris-HCl, pH 6.8) at 100°C for 5 min. Samples were separated by SDS-PAGE (10%). Proteins were transferred to a polyvinylidene difluoride membrane (ATTO, Tokyo, Japan) using a semidry transblot system (ATTO). The blot was blocked for 2 h with 3% BSA/PBS, followed by incubation with 2 µg/ml sheep anti-human CD14 polyclonal Ab (Genzyme/Techne, Minneapolis, MN) in 3% BSA/PBS for 2 h at room temperature. The blot was washed four times with PBS and then incubated for 2 h with HRP-conjugated affinity-purified donkey anti-sheep IgG (Jackson ImmunoResearch, West Grove, PA) at 1:2000 in 3% BSA/PBS. After washing, CD14 was visualized with diaminobenzidine. The Mr of the proteins was estimated by comparison with the position of the standard (Bio-Rad Laboratories, Hercules, CA).
Detection of IL-8 by ELISA
A total of 5 x 104 HGF was cultured
in -MEM with 10% FCS in wells of 24-well collagen I-coated plates
until confluent, and an additional 4-day culture was allowed. After
washing with PBS three times, confluent monolayers of HGF were treated
with 20 µg/ml HLE in HYBRI-MAX for 30 min at 37°C. HLE-treated
monolayer cells were gently washed twice with prewarmed HYBRI-MAX,
followed by addition of test stimulants in 500 µl of HYBRI-MAX
without FCS for 4 h. For the blocking experiment with mAb,
confluent monolayers of HGF were pretreated with 5 µg/ml dialyzed
anti-CD14 mAb (MY4) or isotype control mouse IgG2b at 37°C for 30
min before addition of stimulants. After stimulation, the supernatants
were collected and the level of IL-8 in the supernatants was determined
with a human IL-8 ELISA kit (Endogen, Woburn, MA). IL-8 assays were
performed exactly as instructed by the ELISA manufacturer. The
concentration of IL-8 in the supernatants was determined using the
Softmax data analysis program (Molecular Devices, Menlo Park, CA). Each
sample was assayed in triplicate.
Statistical analysis
All experiments in this study were performed at least three times to test the reproducibility of the results, and representative findings are shown. In some experiments, experimental values are given as means ± SDs. The statistical significance of differences between two means was evaluated by Student’s unpaired t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Confluent monolayers of HGF were cocultured with purified PMNs in
the presence or absence of PMA, and CD14 expression was evaluated by
flow cytometry using the MEM-18 mAb. When HGF were cocultured with
3 x 105 PMNs in the presence of 100 ng/ml
PMA for 1 h, the MFI of CD14 was significantly
(p < 0.01) reduced (MFI = 52% of
control), and 3 x 106 of activated PMNs
caused up to 95% reduction of the CD14 expression (Fig. 1
A). However, unactivated PMNs
lacked the activity at any number of PMNs. Next, we examined whether
the reduction of CD14 expression required secreted molecules from PMNs
or cell-to-cell contact between HGF and PMNs. When the HGF monolayer
was incubated with the culture supernatant derived from the indicated
number of PMA-activated PMNs, CD14 expression on HGF was significantly
(p < 0.01) reduced, and the supernatant from
3 x 106 of activated PMNs reduced the
expression to approximately 75% of the control value (Fig. 1B). When activated PMNs were fixed with 3%
paraformaldehyde, as described previously (20), to prevent
continuous secretion of molecules from PMNs during coculture with the
HGF monolayer, the CD14 expression on HGF was also significantly
(p < 0.01) reduced and 3 x
106 of fixed PMNs reduced to approximately 70%
of the control (Fig. 1C). In addition to PMA stimulation, we
examined whether a physiological stimulus also had a simillar effect.
Fig. 1D shows that when PMNs were stimulated with 1 µM
FMLP for 60 min after priming with 100 ng/ml TNF- for 10 min, the
supernatant from those PMNs showed CD14 cleavage activity, to a degree
approximately 80% that of the PMA supernatant, in a cell
number-dependent fashion. These findings indicate that the reduction of
CD14 expression on HGF was caused by molecules that were both released
and expressed by activated PMNs.
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Since activated PMNs release various proteases and induce the cell
surface-bound serine proteases (20, 21), we next examined
the effect of various protease inhibitors on the reduction of CD14
expression by cell-free supernatant from PMA-stimulated PMNs. A
naturally occurring serine protease inhibitor, 1-AT, and a synthetic
serine protease inhibitor, PMSF, showed a marked inhibitory effect
(83.6% and 79.9%, respectively) on the reduction of CD14 caused by
cell-free supernatant (Table I).
Furthermore, HLE/CMK, a specific inhibitor of HLE, exhibited 62.1%
inhibition of CD14 reduction caused by the supernatants. However, the
inhibitory effect was scarcely observed in 1,10-phenanthroline (a
metalloprotease inhibitor) and E-64 (a cysteine protease inhibitor),
and only slight inhibition (15.9%) was observed in pepstatin (an
asparatic protease inhibitor). These findings indicated that among the
various proteases released from activated PMNs, serine proteases, in
particular HLE, participated in CD14 cleavage caused by the cell-free
supernatant.
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1-AT,
1,10-phenanthroline, E-64, and pepstatin showed almost no inhibitory
effect. These findings indicated that although cell surface serine
proteases actually participated in the reduction of CD14 expression,
the degree was very low. This observation might be due to: 1) the
protease inhibitors were ineffective at cell-to-cell contact
proteolysis, or 2) some protease(s) that was/were not inhibited by the
protease inhibitors used in this study might be involved in the
reduction. No inhibitory effect of 1-AT was supported by the study
of Owen et al. (20), in which cell surface HLE was
resistant to inhibition by naturally occurring protease inhibitors. The
CD14-reducing activity induced by TNF- and FMLP treatment of PMNs
shown in Fig. 1
D was also inhibited by HLE/CMK at
approximately 90% (data not shown). Therefore, the findings shown in
Fig. 1 and Table I indicate that the reduction of CD14 on HGF caused by
activated PMNs was performed mainly by serine proteases, probably HLE
in the supernatant and, although the degree was low, also partially
performed by cell surface HLE. Reduction of CD14 on HGF by HLE treatment
Since the major serine proteases released by activated PMNs are
HLE, cathepsin G, and PR3, we next examined the effect of HLE,
cathepsin G, and PR3 on CD14 expressed by HGF. Fig. 2A shows a representative FACS
profile, indicating that marked reduction of CD14 expression on HGF
treated with 20 µg/ml HLE in the serum-free condition for 1 h
was observed compared with untreated cells. This reduction of CD14 on
HGF reached 85% at 10 µg/ml (340 nM) of HLE, and was totally
achieved at 20 µg/ml (680 nM) of HLE for 60-min treatment (Fig. 2B). In contrast, cathepsin G, which is also inhibited by
1-AT (22) and PMSF (23), and PR3 exhibited
much less activity for CD14 reduction compared with the same
concentration (nM) of HLE. These findings indicated that HLE was the
main serine protease from activated PMNs responsible for CD14 reduction
in the coculture experiment, as shown in Table I. The reduction of CD14
by HLE was inhibited almost completely by pretreatment with 100 µg/ml
1-AT and 10% (v/v) of human serum, which is known as a source of
1-AT (Fig. 2C). The time kinetics experiment revealed
that complete reduction of CD14 was observed after 60-min treatment,
and that even 30-min treatment was adequate to almost completely reduce
CD14 from the cell surface (Fig. 2D).
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stimulation on HGF (19), the HGF monolayer was
stimulated with IL-1 for 5 days before analysis of CD26 sensitivity.
HLE treatment (20 µg/ml) of HGF for 1 h reduced the expression
of CD14 completely, but only slight reduction of CD26, CD59, CD157, and
MHC class I (at most 12%, 28%, 7%, and 24% reduction, respectively)
was observed (Fig. 3), indicating that
HLE preferentially reduced CD14. Next, we examined whether CD14 could
be reexpressed on the cell surface after reduction of CD14 by HLE. CD14
was gradually reexpressed in the normal culture condition, and
approximately 75% of CD14 was recovered within 48 h (Fig. 4).
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These findings suggest that proteolytic cleavage of CD14 on the
cell surface by HLE was the most plausible mechanism, although there
remain two other possibilities that CD14 was internalized or shed by
endogenous enzymes following HGF activation by HLE. To clarify this,
HGF were fixed with 1% paraformaldehyde before HLE treatment. As shown
in Fig. 5, after fixation of HGF, HLE was
still capable of effectively cleaving CD14 (p
< 0.01). Considering that the shedding and the internalization of CD14
should require activating endogenous enzyme(s) or cell activation, this
finding suggests that HLE cleaved CD14 on the cell surface
proteolytically.
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Another approach was to examine the proteolytic cleavage of CD14
by HLE. Purified cell membranes of HGF were treated with HLE, and both
the cell membrane and extracellular supernatant were analyzed by
Western blotting using anti-CD14 polyclonal Ab. As a positive
control, PI-PLC, an enzyme that specifically removes GPI-anchored
proteins from the cell surface (9), was used in this
experiment. In untreated cell membranes, a strong 55-kDa CD14 band was
detected, whereas this band disappeared after HLE treatment and partial
reduction of the band was observed after PI-PLC treatment (Fig. 6). This partial reduction of the CD14
band in the PI-PLC-treated cell membrane was consistent with flow
cytometry in which CD14 expression was partially reduced (MFI, 75.7
(control) vs 28.9 (PI-PLC treatment)). In the extracellular
supernatant, a strong 55-kDa band was detected in the PI-PLC-treated
extracellular supernatant, whereas no bands were detected in
HLE-treated extracellular supernatant or in controls. This finding and
Fig. 5 indicate that reduction of CD14 by HLE resulted from direct
proteolysis, but not from shedding or internalization following cell
activation, and could be degraded into multiple CD14 fragments that
were not detected by polyclonal Ab for CD14.
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The observation that HLE cleaved CD14 on HGF raises the question
whether HLE inhibits LPS-induced IL-8 production by HGF, since it was
reported that HGF produce IL-8 in response to LPS stimulation in a
membrane CD14-dependent pathway (8). Preincubation of HGF
with 20 µg/ml HLE for 30 min markedly suppressed IL-8 production from
HGF upon stimulation with 10 ng/ml LPS (Fig. 7A). Anti-CD14 mAb (MY4)
inhibited LPS (10 ng/ml)-induced IL-8 production by HGF to background
levels, but isotype-matched control Ab did not exhibit any inhibitory
effect. We performed similar experiments with IL-1 as a
CD14-independent stimulant. As shown in Fig. 7B, HLE
pretreatment with HGF did not significantly change IL-8 production
triggered by IL-1. MY4 and isotype-matched Ab were both unable to
inhibit IL-8 production by HGF triggered with IL-1. Thus, these
findings indicate that HLE treatment of HGF specifically inhibited the
CD14-dependent cell activation triggered by LPS.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), which suggested that cathepsin G and PR3 are only
partially involved in the reduction of CD14 expression.
We next focused on the mechanism of the reduction caused by HLE
released from PMNs upon activation. HLE exhibited a dose- and
time-dependent proteolysis of CD14 on the cell surface. As a
consequence of CD14 cleavage, CD14-dependent IL-8 production triggered
by LPS was inhibited. In many cases, the shedding of cell surface
molecules followed cell activation (25, 26, 27). Since HLE
induces cell activation, such as activation of MAP kinase in epithelial
cells (28) and chemokine production by macrophages
(29), the possibility that CD14 reduction is mediated by
cell activation was examined using fixed HGF and purified HGF membrane.
It was clearly demonstrated that reduction of CD14 by HLE was due to
the direct proteolysis of CD14 on the cell surface (Figs. 5 and 6). It
is unlikely that reduction of CD14 expression on HGF resulted from the
direct action of PMA to HGF because no change in CD14 expression on HGF
was observed when the HGF monolayer was stimulated with 100 ng/ml PMA
for 1 h in the absence of PMNs (data not shown). Furthermore,
multiple fragmentation of CD14 might occur, since polyclonal CD14 Ab
did not detect the products by Western blotting analysis. These
findings suggested that CD14 molecules proteolyzed by HLE had no
ability to work as a soluble CD14 (sCD14). CD59 and CD157, other
GPI-anchored glycoproteins expressed on HGF, were not reduced as
efficiently as CD14 by HLE (Fig. 3), which excluded the possibility
that HLE preferentially cleaves GPI-anchored molecules on the cell
surface. Note that the trypsinization itself to harvest HGF did not
affect the CD14 expression. Moreover, when HLE-treated HGF immediately
before confluence was harvested by EDTA without trypsin, CD14
expression was reduced by as much as that by trypsinization (data not
shown). These observations ruled out the possibility that HLE alters
the accessibility of CD14, which is then released at
trypsinization.
Since HLE is induced on the cell surface upon activation (20, 30), this suggested that cell-bound HLE was also involved in the
reduction. To clarify this possibility, fixation of the PMNs was a
suitable procedure, because 1) the fixation does not affect the
enzymatic activity (20, 30), 2) the fixation is necessary
to diminish the tight adhesion of PMNs to HGF since the tight adhesion
can create microenvironments from which inhibitors are excluded
(31, 32), 3) the fixation prevents leakage of endogenous
proteases. However, CD14 reduction by the fixed PMNs was only slightly
inhibited by PMSF and HLE/CMK, and the other protease inhibitors did
not significantly inhibit the reduction (Table I). The lack of an
effect of 1-AT on fixed cell-induced CD14 reduction (Table I) was
consistent with a previous study that reported that cell-bound serine
proteases are remarkably resistant to inhibition by naturally occurring
high m.w. protease inhibitors such as 1-AT (20).
However, low m.w. synthetic inhibitors such as PMSF and HLE/CMK
substantially inhibit cell surface protease activities
(20), and 85% inhibition of cell-bound HLE (fixed PMNs)
activity was shown with 60 µM of HLE/CMK at 5 x
105 fixed PMNs. In the present study, the more
effective condition of 1 mM of HLE/CMK for 3 x
106 of fixed PMNs was used. Therefore, it was
suggested that only slight inhibition of CD14 reduction by PMSF and
HLE/MCK was not due to the resistance of serine proteases against these
inhibitors, and that some protease(s) that was/were not inhibited by
the protease inhibitors used in this study might be involved in the
reduction.
HGF not only functions to support frameworks by synthesis of
extracellular matrix, but also participates in inflammatory and immune
responses to bacterial components such as LPS from
periodontitis-associated Gram-negative bacteria. Activation of HGF via
the LPS-CD14 pathway induces chemokines such as IL-8 (8),
which attract PMNs from the peripheral blood to the inflammatory site
to form a first line host defense, indicating that this mechanism is of
great importance for innate immunity. A previous study reported
(33) that HLE was able to digest IL-8 and abolish the
chemotactic activity, which suggested the possible mechanism of the
down-regulatory cascade of inflammation by HLE. The present study
showed that production of IL-8 was inhibited via CD14 cleavage by HLE,
which might be another mechanism for the down-regulatory cascade of
inflammation by HLE. Since HLE is released from PMNs via the
2 integrin-ICAM-1 pathway (34, 35), and ICAM-1 is cleaved by HLE (36), HLE is
likely to auto-down-regulate HLE production from PMNs possibly by
impairment of the 2 integrin-ICAM-1 pathway,
which is a negative feedback effect of HLE.
HLE is an essential factor for host defense against bacteria
(37), and also has the capacity to degrade a wide variety
of extracellular matrix including elastin, proteoglycan, denatured
collagen, fibronectin, and laminin (38, 39), which are
characteristics of several pathological conditions, including
periodontitis (2, 3, 4), rheumatoid arthritis, respiratory
distress syndromes, and blistering skin diseases (5, 6).
Recently, HLE was reported to affect on the various functions of
resident nonhemopoietic cells and hemopoietic immune cells by
proteolytic activity. For example, HLE treatment results in the release
of basic fibroblast growth factor and heparan sulfate proteoglycans
from pulmonary fibroblasts (40, 41) and the release of
TGF1 from epithelial and endothelial cells (42), all of
which could influence the tissue repair mechanism. In immune cells,
specific leukocyte Ags, such as CD2, CD4, CD8 (43), CD43
(44), and CD54 (36), are cleaved by HLE.
Recently, treatment of monocytes with HLE resulted in CD14 reduction,
followed by inhibition of TNF- production triggered by LPS
(45), and it was demonstrated that sCD14 can be
proteolyzed into multiple fragments by HLE. Considering that no
CD14-expressing HGF are activated with LPS in a serum-derived
sCD14-dependent manner (46), HGF activation with LPS could
be inhibited by HLE in a sCD14-dependent manner as well as a cell
surface CD14-dependent manner.
It is unclear whether reduction of CD14 on HGF by HLE could also occur
in vivo. Previous studies demonstrated that activated PMNs may release
up to 380 nM HLE into the extracellular medium (47), and
the number of PMNs may increase by even 100-fold at the inflammatory
site (48, 49). In addition, although serum contains
abundant naturally occurring protease inhibitors, pericellular
concentrations of HLE exceed that of naturally occurring inhibitors by
approximately 2 orders of magnitude (31, 32), resulting in
an escape from the action of protease inhibitors. Reduction of CD14 on
HGF by HLE released from activated PMNs under the coculture condition
shown in the present study (Fig. 1) was close to the in vivo condition.
Considering the present findings and those of previous studies,
reduction of CD14 on HGF caused by activated PMNs is likely to occur
in vivo.
In conclusion, the present study demonstrated that direct interaction between HGF and PMNs regulated the inflammatory responses induced by LPS. This mechanism could exist in physiological conditions as a new negative feedback mechanism of inflammation. Early-onset periodontitis is characterized by severe, rapidly progressive, early-onset alveolar bone loss and familial aggregation (50). PMNs from early-onset periodontitis patients have been reported to produce much higher HLE than controls (51). This may lead to an altered physiological balance regulated by the negative feedback mechanism shown in the present study. The present findings suggest that interaction of HGF and PMNs leading to the regulation of inflammatory responses may provide an additional viewpoint to understand the mechanism of onset and development of inflammatory diseases such as periodontitis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eiji Nemoto, Division of Periodontics and Endodontics, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; 1-AT, 1-antitrypsin; HGF, human gingival fibroblast; HLE, human leukocyte elastase; HLE/CMK, N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone; MFI, mean fluorescence intensity; PI-PLC, phosphatidylinositol-phospholipase C; PR3, proteinase 3; sCD14, soluble CD14.
Received for publication March 30, 2000. Accepted for publication August 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 from the extracellular matrix of cultured human epithelial and endothelial cells. J. Biol. Chem. 270:4689.This article has been cited by other articles:
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