Division of Periodontics and Endodontics, Tohoku University Graduate School of Dentistry, Sendai, Japan
Correspondence: Dr. Eiji Nemoto, Division of Periodontics and Endodontics, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. E-mail: eiji{at}mail.cc.tohoku.ac.jp
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, suggesting that HLE inhibited a
CD40-dependent cell activation. These results suggest that HLE
possesses an anti-inflammatory effect for the HGF-mediated inflammatory
process.
Key Words: neutrophils • periodontitis • inflammation • cell surface molecules • serine protease
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CD40 is a 50-kDa membrane-bound type I glycoprotein, which is a member
of the tumor necrosis factor (TNF-) receptor superfamily
described initially on B lymphocyte, where it binds to a CD40 ligand
(CD40L; also known as gp39, CD154) on activated T lymphocytes and plays
a critical role in B cell activation and isotype switching
[4
]. CD40 is subsequently expressed on non-B lymphocyte
lineage cells [4
, 5
] such as monocytes,
macrophages, DC, epithelial cells, endothelial cells, smooth muscle
cells, and fibroblasts. Engagement of CD40 on fibroblasts results in
up-regulation of costimulatory and adhesion molecules such as
intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion
molecule-1 [6
] and production of proinflammatory
cytokines/chemokines [7
, 8
], interleukin
(IL)-1, IL-6, and IL-8, vascular endothelial cell growth factor
[9
], cyclooxygenase-2, prostaglandin E2
[10
], and extracellular matrix such as hyaluronate
[11
]. Furthermore, CD40 on fibroblasts costimulate T
lymphocyte proliferation, which is independent of B7-1 and -2
[7
]. In periodontal tissue as well, the involvement of
CD40 on human gingival fibroblasts (HGF) with inflammation was
demonstrated by in vitro and in vivo studies [8
,
12
]. Thus, the CD40-CD40L system is an important pathway
for the fibroblast/immune system.
In acute inflammatory reactions, multiple chemoattractants regulate
polymorphonuclear leukocyte (PMN) trafficking [13
], and
PMN migrate across blood vessel endothelial cells and then move toward
inflammatory lesions through connective tissue, which is composed of
mainly fibroblasts and extracellular matrix. Infiltration,
accumulation, and retention of PMN in connective tissues are common
characteristics of inflammation. PMN are the first line of host defense
against mucosal infection, particularly human leukocyte elastase (HLE),
a serine protease that is stored in the azurophile granules and is an
essential factor for host defense against bacterial infection
[14
]. However, most studies have focused on the effects
of HLE with its detrimental potential for enzymes as a result of its
extracellular matrix-degrading activity [15
,
16
]. As a consequence of extensive proteolysis of
extracellular matrix, HLE has been reported to be associated with the
pathogenesis of various inflammatory diseases such as cystic fibrosis,
acute respiratory distress syndrome, rheumatoid arthritis, pulmonary
emphysema, bronchitis, and periodontitis [15
,
16
]. Recently, however, HLE was suggested to have
potential immunoregulatory functions [17
] via cleavage
of cell surface molecules such as CD2, CD4, and CD8 [18
]
on lymphocytes; CD16 [19
] and CD43 [20
]
on PMN; ICAM-1 [21
] on hemotopoietic cell lines; and
CD14 on monocytes [22
] and fibroblasts
[23
]. Furthermore, the cytokine network is controlled by
proteolysis of cytokines such as TNF- [24
], IL-2
[25
], IL-6 [26
], and IL-8
[27
] and cytokine receptors such as TNF receptor (75
kDa) [28
], IL-2 receptor [29
], and IL-6
receptor [29
] by HLE, which result in cytokine
inactivation or prevention of cellular responses to cytokines.
The potential, immunoregulatory function of HLE in local inflammatory processes led us to examine the sensitivity of CD40 expressed by HGF for this enzyme.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was obtained from Dainippon Pharmaceutical Co. Ltd. (Osaka,
Japan). Human natural interferon-
(IFN-; antiviral activity,
8.0x106 IU/mg protein) was provided by the Hayashibara
Bioscience Institute (Okayama, Japan). -Minimum essential medium
(MEM) and 0.25% trypsin-1 mM ethylenediaminetetraacetate (EDTA) were
from Gibco-BRL (Rockville, MD). Anti-CD10 [HI10a, mouse immunoglobulin
G (IgG)1], anti-CD73 (AD2, mouse IgG1), and anti-CD26 monoclonal
antibody (mAb; M-A261, mouse IgG1) were from PharMingen (San Diego,
CA). Anti-CD40 mAb (EA-5, mouse IgG1) was from BioSource International
(Camarillo, CA). All isotype-control mAb for fluorescein-activated cell
sorter (FACS) were obtained from Immunotech (Marseille, France). All
other reagents were obtained from Sigma Chemical Co. unless otherwise
indicated.
Fibroblasts
HGF was prepared from the explants of normal gingiva from 8- to
25-year-old patients with informed consent, as reported previously
[30
]. 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% fetal calf
serum (FCS; Flow Laboratories, McLean, VA) 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
phosphate-buffered saline (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. Human skin fibroblasts
(SF-MA) and human lung fibroblasts (MRC-5) were obtained from the
Japanese Cancer Research Resources Bank (Tokyo, Japan). Human skin
fibroblast (FS-4) was supplied by M. Kohase, National Institute of
Infectious Diseases (Tokyo, Japan). These fibroblasts were maintained
in -MEM supplemented with 10% FCS.
Isolation of peripheral blood mononuclear cell (PBMC)-derived
monocytes, macrophages, and mature DC
PBMC from heparinized (10 U/ml) peripheral venous blood were
isolated by density gradient centrifugation on Ficoll-Paque PLUS®
(Amersham Biosciences Inc., Piscataway, NJ) at 400 g for 30
min at room temperature. The fraction containing PBMC 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. Monocytes
were separated from lymphocytes by adherence to plastic dishes.
Briefly, PBMC suspended in RPMI 1640 containing 20% FCS were incubated
for 1 h at 37°C in six-well multiplates (4x106
cells/well). After removal of nonadherent cells by washing with
prewarmed PBS three times, monocytes were harvested by treatment with
Cell Dissociation Solution®. To allow monocytes to differentiate to
macrophages, adherent monocytes were cultured in RPMI 1640 containing
15% FCS for 7 days as described previously [31
].
Nonadherent and adherent macrophages were recovered by centrifugation
of cell suspension and treatment with Cell Dissociation Solution®,
respectively. Both macrophages were mixed together and used as
PBMC-derived macrophages. For induction of DC, adherent cells in
six-well multiplates were incubated in RPMI 1640 containing 5% FCS for
7 days in the presence of 500 U/ml rhIL-4 and 800 U/ml rhGM-CSF. To
allow DC to differentiate to mature DC, DC were stimulated with 1
µg/ml E.coli LPS in RPMI 1640 containing 5% FCS for 2
days [32
].
HLE treatment
Monolayers of fibroblasts in 24-well multiplates [the well
contained 300 µl PBS containing 0.1% (w/v) bovine serum albumin
(BSA)] were treated with the indicated concentration of HLE at 37°C
for the indicated times. Monocytes, macrophages, and DC
(2x105 of each) suspended in 300 µl PBS containing 0.1%
(w/v) BSA were treated with 680 nM (20 µg/ml) HLE at 37°C for
1 h. For pretreatment of HLE with HLE inhibitors, 680 nM HLE was
preincubated with 1 mM PMSF or 10% (v/v) human serum for 15 min at
37°C before addition onto monolayer cells.
Flow cytometry
HGF was collected by Cell Dissociation Solution®, washed three
times with PBS, and used for staining. HGF (105 cells) were
stained with each mAb or each isotype-matched control IgG at 4°C for
20 min. Following washing, fluorescein isothiocyanate-conjugated goat
anti-mouse IgG (BioSource International) was added at 4°C for 20 min.
For CD40L binding, HGF (105 cells) was incubated for 45 min
at 4°C with 80 µl hCD40L-muCD8 (Ancell Co., Bayport, MN) at a
concentration of 5 µg/ml. Cells were washed twice and incubated with
anti-mouse CD8 (53-6.7, rat IgG2a) conjugated with
phycoerythrin (PE; eBioscience, San Diego, CA),
after which they were washed three times. Staining was analyzed on a
FACScan® (Becton Dickinson, Mountain View, CA). Measurements 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).
For staining-fixed HGF, monolayers of HGF in 24-well multiplates were
treated with 1% (w/v) paraformaldehyde for 5 min at room temperature.
After being washed three times with PBS, HGF was treated with HLE as
described above and was harvested by Cell Dissociation Solution® and
then stained with anti-CD40 mAb.
Preparation of cell membranes and cell lysates
To prepare cell membranes, HGF was collected by Cell
Dissociation Solution®, and cells were suspended in hypotonic buffer
(10 mM Tris-HCl, pH 7.4, and 1 mM MgCl2) and were 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 g for 5 min twice
to remove nuclei. Supernatants were centrifuged at 15,000 g
for 30 min, and membrane pellets were suspended in PBS and stored at
-20°C until use. To prepare cell lysates, HGF (cells from confluent
HGF grown in a 6.25 cm2 area/20 µl) was suspended in PBS
containing 1% Nonidet P-40 and 1 mM PMSF and was incubated on ice for
30 min followed by centrifugation at 12,000 g at 4°C for
10 min; then, supernatants were collected and stored at -20°C until
use.
Western blotting
Cell membranes from confluent HGF grown in a 12.5
cm2 area were suspended in 20 µl Hanks’ balanced salt
solution (HBSS) containing 1 µg HLE and were incubated for the
indicated times at 37°C. Samples of cell membranes (20 µl) and cell
lysate (20 µl) were solubilized with 10 µl Laemmli sample buffer
[20% glycerol, 4% sodium dodecyl sulfate (SDS), 0.1%
2-mercaptoethanol, 0.002% bromophenol blue, and 120 mM Tris-HCl, pH
6.8] at 100°C for 5 min. Samples were separated by
SDS-polyacrylamide gel electrophoresis (PAGE; 10%). Proteins were
transferred to a polyvinylidene difluoride (PVDF) membrane (ATTO Co.,
Tokyo, Japan) using a semidry transblot system (ATTO). The blot was
blocked with 5% (w/v) nonfat dried milk, 0.1% (v/v) Tween 20, in PBS
at 4°C overnight followed by incubation with 0.3 µg/ml goat
anti-human CD40/TNFRSF5 polyclonal Ab (R&D Systems Inc., Minneapolis,
MN) in 0.1% Tween 20 in PBS for 2 h at room temperature. The blot
was washed four times with 0.1% Tween 20 in PBS and then incubated
with horseradish peroxidase-conjugated affiniPure donkey anti-goat IgG
(Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at 1:50,000
in 0.1% Tween 20 in PBS for 2 h at room temperature. After
washing, the blot was treated with Western blotting detection reagent
ECL Plus® (Amersham Pharmacia Biotech Inc., Piscataway, NJ) to
produce a chemiluminescence as instructed by the manufacturer. The
detected blot was exposed to PolaroidTM film using the ECL mini-camera.
The molecular weight of the proteins was estimated by comparison with
the position of the standard (Bio-Rad Laboratories, Hercules, CA).
Detection of IL-8 and monocyte chemoattractant protein-1
(MCP-1) by enzyme-linked immunosorbent assay (ELISA)
HGF was cultured in -MEM with 10% FCS in wells of
96-well collagen I (rat-tail tendon)-coated plates (Becton Dickinson
Labware) until confluent and was stimulated with 1000 U/ml IFN- for
3 days. After being washed with PBS three times, confluent monolayers
of HGF were treated with 680 nM HLE in PBS containing 0.1% (w/v) BSA
for 15 min at 37°C. HLE-treated monolayer cells were gently washed
twice with prewarmed -MEM, followed by addition of test stimulants
in 100 µl -MEM with 5% FCS for 6 h. After stimulation, the
supernatants were collected, and the level of IL-8 and MCP-1 in the
supernatants was determined with human IL-8 ELISA kits (Endogen Inc.,
Woburn, MA) and OptEIATM human MCP-1 ELISA kits (PharMingen),
respectively. The assays were performed exactly as instructed by the
ELISA manufacturer. The concentration of IL-8 and MCP-1 in the
supernatants was determined using the Softmax data analysis program
(Molecular Devices Co., Menlo Park, CA). Each sample was assayed in
triplicate.
Statistical analysis
All experiments in this study were performed at least two or
three times to test the reproducibility of the results, and
representative findings are shown. In some experiments, experimental
values are given as means ± SE. The statistical
significance of differences between two means was evaluated by
Student’s unpaired t-test.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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treatment [5
], we used
untreated and IFN--treated HGF for HLE treatment. In Figure 1A
and B, representative FACS profiles of untreated and
IFN--treated HGF, respectively, showed that approximately two- to
threefold higher expression of CD40 was induced by IFN- stimulation
as evaluated by MFI, and significant reduction of CD40 expression on
untreated and IFN--treated HGF by 680 nM HLE treatment for 1 h
was observed compared with HLE-untreated cells. We examined whether the
binding of CD40L to HGF should be reduced by HLE treatment. HGF,
untreated or treated with IFN-, was treated with 680 nM HLE and then
incubated with CD40L fusion protein, followed by staining for flow
cytometry. Figure 1C
and 1D
, shows representative FACS profiles,
indicating that untreated or IFN--treated HGF binds to CD40L, and
HGF treated with IFN- displays approximately twofold higher binding
to CD40L as evaluated by MFI. Binding of CD40L to untreated and
IFN--treated HGF was completely diminished by HLE treatment for
1 h, respectively (Fig. 1C
and 1D)
. The time kinetics experiments
revealed that almost complete reduction of binding of CD40L to
untreated and IFN--treated HGF was observed only after a 7- to
15-min treatment. On the other hand, CD40 expression was reduced only
slightly at this time point (Fig. 1E
and 1F)
. The reduction of CD40 on
both HGF samples reached about 50% after a 60-min treatment and
gradually decreased to 30–40% of CD40 expression after a 120-min
treatment (Fig. 1E
and 1F)
.
 View larger version (54K): [in a new window] |
Figure 1. Reduction of CD40 expression on HGF and CD40L binding to HGF by HLE
treatment. HGF monolayer cells were cultured with IFN- (1000 U/ml)
stimulation (B, D, and F) or without (A, C, and E) for 3 days and were
then treated with 680 nM HLE for the indicated time at 37°C. After
treatment, cells were harvested using Cell Dissociation Solution® and
stained with EA-5, matched-isotype Ab, or CD40L protein and were
analyzed by flow cytometry. (A and B) A representative FACS profile of
CD40 expressed on HGF after treatment with or without 680 nM HLE for
1 h. (C and D) A representative FACS profile of CD40L binding to
HGF after treatment with or without 680 nM HLE for 1 h. (E and F)
HGF was treated with 680 nM HLE for the indicated time, and then CD40
expression and CD40L binding to HGF were analyzed. Findings are
representative of three independent experiments.
|
-treated
HGF was reduced by 340 nM HLE treatment for 1 h (Fig. 2 A
), and additional, slight reductions of CD40 were observed by 680
nM HLE treatment in both HGF samples. As HLE is one of the serine
proteases, we examined the effect of CatG (EC 3.4.21.20) and PR3 (EC
3.4.21.76), HLE homologues of serine proteases, on CD40 expressed by
HGF. Figure 2B
shows that in contrast to HLE, CatG and PR3 exhibited
much less activity for CD40 reduction compared with the same
concentration of HLE. The reduction of CD40 by HLE was inhibited
completely by pretreatment with 10% (v/v) human serum, which is known
as a source of natural protease inhibitors such as 1-antitrypsin
[33
] and with 1 mM PMSF, a serine protease inhibitor
(Fig. 2C)
, indicating that enzymatic activity was required for the
reduction.
 View larger version (18K): [in a new window] |
Figure 2. Reduction of CD40 expression on HGF by HLE treatment. HGF monolayer
cells were cultured with no stimulation or with IFN- (1000 U/ml) for
3 days and were then treated with the indicated concentrations of HLE
for 1 h at 37°C. After treatment, cells were harvested and
stained with EA-5 or matched-isotype Ab and analyzed by flow cytometry.
(A) HGF stimulated with or without IFN- was treated with the
indicated concentrations of HLE. (B) HGF stimulated with IFN- was
treated with the indicated concentrations of HLE, CatG, or PR3 for
1 h. (C) HGF stimulated with IFN- was treated with 680 nM HLE
for 1 h in the presence or absence of 10% (v/v) human serum
or 1 mM PMSF. Representative findings of three independent experiments
are shown as the mean ± SE of duplicate assays.
|
 View larger version (53K): [in a new window] |
Figure 3. Effect of HLE treatment on ectoenzyme expressed by HGF. HGF monolayer
cells were treated with 680 nM HLE for 1 h, and harvested cells
were examined for CD40, CD10, CD26, and CD73 expression by flow
cytometry. HGF was stimulated with IFN- (1000 U/ml) for 3 days or
IL-1 (10 ng/ml) for 5 days to induce CD40 or CD26
[30
], respectively, before HLE treatment.
Representative findings of three independent experiments are shown as
the mean ± SE of duplicate assays. Statistical
significance is shown (*, P<0.01 vs. untreated HGF).
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View this table: [in a new window] |
Table 1. CD40 Expression and HLE Sensitivity of CD40 Expressed by Human
Fibroblasts and Myeloid Lineage
Cellsa
|
 View larger version (17K): [in a new window] |
Figure 4. Cleavage of CD40 on fixed HGF and on HGF membrane by HLE treatment. (A)
IFN--stimulated HGF monolayer cells were fixed with 1%
paraformaldehyde for 5 min at room temperature. After washing, unfixed
or fixed cells were incubated with 680 nM HLE for 1 h at 37°C.
Expression of CD40 on HGF was assessed by flow cytometry. (B) HGF was
collected by Cell Dissociation Solution®. Cells from confluent HGF
grown in 6.25 cm2 area were suspended in 20 µl HBSS
containing 1 µg HLE and incubated for 1 h at 37°C.
Supernatants were separated from cell pellets by centrifugation. (C)
Purified cell membrane of HGF was treated with HLE for the indicated
time at 37°C. Supernatants (Sup) with/without HLE treatment, control
cell lysate (Cont.), and cell lysates (Cell) with/without HLE treatment
(B) and cell membranes (C) were solubilized with Laemmli sample buffer,
subjected to 10% SDS-PAGE, and transferred to PVDF membrane. The blot
was probed with an anti-CD40 polyclonal Ab. Molecular mass markers
(kDa) are shown on the left. Representative findings of three
independent experiments are shown as the mean ± SE of
duplicate assays (A), and two independent experiments are shown (B and
C). Statistical significance is shown (*, P<0.01 vs.
respective control).
|
produce
cytokines/chemokines in response to CD40L stimulation, and this
production was up-regulated by IFN- treatment before stimulation
[7
, 8
]. Therefore, the observation that HLE
cleaved CD40 on HGF raises the question whether HLE inhibits
CD40L-induced IL-8 and MCP-1 production by HGF. CD40L stimulation
induced IL-8 and MCP-1 production from IFN--treated HGF in a
dose-dependent manner (Fig. 5A
and B), but significant production was not detected
from non-IFN--treated HGF (data not shown). Therefore, this series
of experiments in Figure 5
was performed using IFN--treated HGF.
Preincubation of IFN--treated HGF with 680 nM HLE for 15 min
markedly suppressed IL-8 and MCP-1 production from HGF upon stimulation
with CD40L (Fig. 5A
and 5B)
. We conducted a similar experiment with
IL-1 as a CD40-independent stimulant. As shown in Figure 5C
and 5D ,
HLE pretreatment with HGF did not significantly change IL-8 and MCP-1
production triggered by IL-1 (P<0.01). Thus, these
findings suggest that HLE treatment of HGF specifically inhibited the
CD40-dependent cell activation triggered by CD40L and did not affect
the function of IL-1 receptor.
 View larger version (53K): [in a new window] |
Figure 5. HLE-treatment of HGF resulted in the reduction of IL-8 and MCP-1
production by HGF in response to CD40L. HGF monolayer cells were
treated with 680 nM HLE for 15 min at 37°C before stimulation with
the indicated concentrations of CD40L (A and B) or 10 ng/ml IL-1 (C
and D) for 6 h at 37°C. The amount of IL-8 (A and C) and MCP-1
(B and D) in the supernatants was analyzed by ELISA. Representative
findings of three independent experiments are shown as the mean ±
SE of triplicate assays. Statistical significance is shown
(*, P<0.01 vs. respective control).
|
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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stimulation. We demonstrated that CD40 on fibroblasts from skin
and lung as well as gingiva was cleaved by HLE, suggesting that this
feedback mechanism by HLE exists on various tissues where fibroblasts
are involved in the inflammatory response. However, this feedback
activity of HLE with CD40 cleavage appears to be specific to the
fibroblast-mediated inflammatory process since CD40 expressed by
myeloid cells, such as monocytes, macrophages, and DC was resistant to
HLE. Although the mechanism that alters the sensitivity for HLE is
currently unclear, post-translational modifications and interactions of
other proteins, such as the cell-specific glycosylation pattern
[39
], expression of membrane protease inhibitor for HLE,
expression of HLE receptor [40
], presence of other HLE
substrate, or complexation with some binding protein as observed
in IL-6 receptor cleavage [26
], may affect the
efficiency of CD40 cleavage at the cell surface. We found that CD10, CD26, and CD73 were relatively resistant to cleavage by HLE. CD10/NEP, CD26/DPPIV, and CD73/5'-NT, all of which are ectoenzymes, are widely expressed on hematopoietic and nonhematopoietic cells [34 ]. CD10/NEP hydrolyzes a variety of physiological-active peptides, such as chemotactic peptide [f-met-leu-phe (fMLP); ref. 34 ]. CD26/DPPIV [30 , 34 ] participates in hydrolysis of several chemokines resulting in abrogation of chemotactic activity [35 ]. CD73/5'-NT catalyzes the extracellular dephosphorylation of nucleoside monophosphates to corresponding nucleosides [34 ] such as adenosine, which inhibits proinflammatory cytokine production [36 ] and superoxide generation [37 ] and induces anti-inflammatory cytokine production [38 ]. In other words, these cell surface molecules have anti-inflammatory properties. Therefore, the relative resistance of these molecules for HLE cleavage among cell surface molecules is consistent with the concept that HLE has a potential feedback activity in the inflammatory response.
HLE, CatG, and PR3, serine proteases localized in the azurophilic granules of neutrophils, share a large sequence homology and have many similar aspects such as processing and granular sorting of enzymes and comparatively broad substrate specificity [41 , 42 ]. In the present study, however, in contrast to HLE, CatG and PR3 exhibited markedly less activity for CD40 cleavage, as in the case of CD14 on HGF reported previously [23 ], which conflicts with the prevailing notion that these enzymes lack specificities and supports the finding that these enzymes have specific and restricted effects on distinct cytokines, cytokines receptors, and cell surface molecules [17 ].
Soluble CD40 (sCD40) has been demonstrated in the supernatant of B cell
lines as a functionally active form [43
] and was also
detected in human serum [44
]. It has been suggested that
sCD40 regulates the CD40-CD40L interaction in a negative fashion
[45
]. In the present study, Western blot analysis (Fig. 4)
showed that CD40 on HGF may be degraded into multiple fragments by
HLE. Therefore, CD40 could be inactivated as in the case of IL-6
[26
], IL-8 [27
], and TNF-
[24
], and it is unlikely that the fragment cleaved by
HLE works as an antagonist as reported in cases of IL-2
[25
], IL-2 receptor [29
], and IL-6
receptor [29
].
We observed a discrepancy between CD40 expression and CD40L binding after HLE treatment in the time kinetics experiments (Fig. 1E and 1F) . This discrepancy could be explained by the previous finding that the epitope recognized by EA-5 (anti-CD40 mAb) is different from the binding site for CD40L [46 ], and this discrepancy suggested that the CD40L binding site was located closer to the N-terminus in the extracellular domain than the EA-5 binding site. It is also possible that there might be a cofactor involved in the binding between CD40 and CD40 ligand that is more sensitive to elastase than is CD40 itself. In this experiment, only several minutes of treatment by HLE was adequate to diminish the binding of CD40L. However, it is unclear whether reduction of binding of CD40L by HLE could occur in vivo, as human serum inhibited the reduction of CD40 by HLE completely (Fig. 2C) . As for the local inflammatory site, the number of PMN may increase by even 100-fold [47 , 48 ], the pericellular concentration of HLE exceeds that of protease inhibitors by approximately two orders of magnitude [49 , 50 ], and released HLE can rebind to the PMN surface [33 ], allowing them not only to be locally concentrated but also to be resistant to naturally occurring inhibitors [33 ]. Moreover, tight adhesion of PMN to HGF may create microenvironments from which inhibitors are excluded [49 , 50 ]. Therefore, the reduction of binding of CD40L caused by HLE is likely to occur in vivo.
Activated T lymphocyte expressing CD40L might be important in triggering the HGF-CD40 pathway in periodontitis, as the T lymphocyte-HGF interaction is reported to be a critical event on periodontitis [51 ]. This CD40/CD40L interacton could activate HGF to produce cytokines/chemokines such as IL-8 and MCP-1, which attract PMN, monocytes, and T lymphocytes from the peripheral blood to the inflammatory site [13 ], and also could costimulate T lymphocytes to proliferate [7 ], suggesting an important mechanism for host defense against infection with microorganisms. However, the excessive inflammatory responses in which the overproduction of cytokines/chemokines interrupts the smooth transition from acute inflammation to acquired immune responses should be controlled to maintain the physiological balance. The present finding that prevention of chemokine production from HGF via CD40 cleavage by HLE suggests a natural feedback mechanism in an inflammatory process. Thus, HLE is a part of the physiological repertoire to control cell-cell interactions and cytokine-cytokine receptor interactions, especially in the PMN-dominated, inflammatory process. However, an excessive production of HLE as observed in severe periodontitis [52 ], prolonged activity of HLE, or impairment of HLE inhibitors may disorder the balance of cell-cell interactions, the cytokine network, and wound healing. Moreover, it could cause severe tissue destruction, which may be associated with the pathogenesis of periodontitis and other various disease.
Finally, the present study may provide another way of understanding the mechanism of onset and development of various inflammatory diseases as well as conquering the disorders induced by excessive production of HLE.
Received February 22, 2002; revised April 16, 2002; accepted April 18, 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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