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(Journal of Leukocyte Biology. 2002;72:538-545.)
© 2002 by Society for Leukocyte Biology

Disruption of CD40/CD40 ligand interaction with cleavage of CD40 on human gingival fibroblasts by human leukocyte elastase resulting in down-regulation of chemokine production

Eiji Nemoto, Hiroyuki Tada and Hidetoshi Shimauchi

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD40 is a crucial element in the process of fibroblast activation. We demonstrated that treatment of human gingival fibroblast (HGF) with human leukocyte elastase (HLE), a neutrophil serine protease, down-regulated the expression of CD40 and binding to the CD40 ligand (CD40L) using flow cytometry. The other neutrophil serine proteases, cathepsin G and proteinase 3, exhibited markedly less activity for CD40 reduction. The CD40 reduction by HLE was also observed in skin and lung fibroblasts, but not in monocytes, macrophages, and dendritic cells. The reduction resulted from direct proteolysis by HLE on the cell surface, because HLE reduced CD40 on fixed HGF and also on cell lysates and membranes. HLE treatment of HGF decreases interleukin (IL)-8 and macrophage chemoattractant protein-1 production by HGF when stimulated by CD40L, but not by IL-1{alpha}, 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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fibroblasts have previously been considered important connective tissue cells that construct a supporting framework crucial for tissue integrity and repair. Furthermore, fibroblasts from different anatomical regions display characteristic phenotypes and are not a homogeneous population even within a single tissue but exist as subsets of cells much like tissue macrophages and dendritic cells (DC) [1 , 2 ]. Recently, fibroblasts were found to be important, sentinel cells in the immune system. Fibroblasts actively define the structure of tissue microenvironments and regulate infiltrated hematopoietic cell functions by production of cytokines/chemokines and extracellular matrix in which system CD40 is a crucial element in the process of fibroblast activation [1 , 3 ].

CD40 is a 50-kDa membrane-bound type I glycoprotein, which is a member of the tumor necrosis factor {alpha} (TNF-{alpha}) 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-{alpha} [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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
HLE (EC 3.4.21.37) and human neutrophil cathepsin G (EC 3.4.21.20; CatG) were purchased from Calbiochem-Novabiochem Co. (La Jolla, CA). Human leukocyte proteinase 3 (EC 3.4.21.76; PR3) was purchased from HyTest (Turku, Finland). Lipopolysaccharide (LPS) of Escherichia coli O55:B5, Cell Dissociation Solution® (nonenzymatic), and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human (rh) IL-4, rh granulocyte-macrophage colony-stimulating factor (GM-CSF), and rh-soluble CD40L/TRAP was obtained from Peprotech EC Ltd. (London, UK). rhIL-1{alpha} was obtained from Dainippon Pharmaceutical Co. Ltd. (Osaka, Japan). Human natural interferon-{gamma} (IFN-{gamma}; antiviral activity, 8.0x106 IU/mg protein) was provided by the Hayashibara Bioscience Institute (Okayama, Japan). {alpha}-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 {alpha}-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 {alpha}-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{alpha} (Ancell Co., Bayport, MN) at a concentration of 5 µg/ml. Cells were washed twice and incubated with anti-mouse CD8{alpha} (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 {alpha}-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-{gamma} 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 {alpha}-MEM, followed by addition of test stimulants in 100 µl {alpha}-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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of HLE treatment on CD40 expressed by HGF and binding of CD40L to HGF
The effect of HLE on CD40 expressed by HGF was evaluated by flow cytometric analysis. As it was reported that CD40 expression on HGF is up-regulated by IFN-{gamma} treatment [5 ], we used untreated and IFN-{gamma}-treated HGF for HLE treatment. In Figure 1A and B, representative FACS profiles of untreated and IFN-{gamma}-treated HGF, respectively, showed that approximately two- to threefold higher expression of CD40 was induced by IFN-{gamma} stimulation as evaluated by MFI, and significant reduction of CD40 expression on untreated and IFN-{gamma}-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-{gamma}, 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-{gamma}-treated HGF binds to CD40L, and HGF treated with IFN-{gamma} displays approximately twofold higher binding to CD40L as evaluated by MFI. Binding of CD40L to untreated and IFN-{gamma}-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-{gamma}-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) .



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  		Figure 1. Reduction of CD40 expression on HGF and CD40L binding to HGF by HLE treatment. HGF monolayer cells were cultured with IFN-{gamma} (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.

 
Requirement of enzymatic activity for CD40 reduction by HLE treatment
The concentration kinetics experiments revealed that approximately half the amount of CD40 expressions on untreated and IFN-{gamma}-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 {alpha}1-antitrypsin [33 ] and with 1 mM PMSF, a serine protease inhibitor (Fig. 2C) , indicating that enzymatic activity was required for the reduction.



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  		Figure 2. Reduction of CD40 expression on HGF by HLE treatment. HGF monolayer cells were cultured with no stimulation or with IFN-{gamma} (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-{gamma} was treated with the indicated concentrations of HLE. (B) HGF stimulated with IFN-{gamma} was treated with the indicated concentrations of HLE, CatG, or PR3 for 1 h. (C) HGF stimulated with IFN-{gamma} 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.

 
Effect of HLE treatment on ectoenzymes expressed by HGF and CD40 expressed by various types of cells
Many substrates, which have been reported to be cleaved by HLE (see the Introduction) share common properties that they are involved in the induction of an inflammatory response. Therefore, we examined whether ectoenzymes with anti-inflammatory properties such as CD10/neutral endopeptidase 24.11 (NEP) [34 ], CD26/dipeptidylpeptidase IV (DPPIV) [35 ], and CD73/5'-nucleotidase (NT) [36 37 38 ] could be substrates for HLE. Figure 3 shows that HLE treatment for 1 h caused only a slight reduction in CD10 and CD26 and no effect on CD73, in contrast to CD40, whose expression was reduced by 65%, indicating that there appears to be selectivity for the cleavage among cell surface proteins. As CD40 is expressed on fibroblasts from various tissues [5 ] and on many cell types (see the Introduction), we tested whether CD40 on fibroblasts of other origins, and distinct cell types have different sensitivities to HLE treatment. Table 1 shows that fibroblasts from gingiva, lung, and skin expressed CD40 at similar levels ranging from 30.6 to 60.3 (MFI), and HLE treatment of these fibroblasts caused CD40 reduction in these cells at similar levels ranging from 48.1% to 69.1% reduction. Furthermore, CD40 expression on myeloid lineage cells from peripheral blood was investigated. Monocytes and macrophages expressed CD40 at similar levels to fibroblasts, but DC expressed CD40 approximately tenfold higher than that of HGF. HLE treatment of these myeloid cells, in contrast to fibroblasts, did not reduce the expression but rather enhanced CD40, particularly in DC, suggesting that CD40 reduction was dependent on cell types.



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  		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-{gamma} (1000 U/ml) for 3 days or IL-1{alpha} (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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  		Table 1. CD40 Expression and HLE Sensitivity of CD40 Expressed by Human Fibroblasts and Myeloid Lineage Cellsa

 
Reduction of CD40 by HLE resulted from direct proteolysis, not shedding or internalization
The reduction probably resulted from proteolytic cleavage on the cell surface by HLE. However, there remained two other possible mechanisms by which CD40 was internalized or shed by endogenous enzymes following HGF activation by HLE. Therefore, HGF was fixed with paraformaldehyde before HLE treatment to clarify this. Figure 4 A shows that HLE treatment reduced CD40 on fixed HGF as well as unfixed HGF. These findings suggested that HLE reduced CD40 on the cell surface proteolytically. Another approach was to examine the proteolytic cleavage of CD40 by HLE. HGF was treated with or without HLE for 1 h, and cell lysates and supernatants were analyzed by Western blotting using anti-CD40 polyclonal Ab. A 50-kDa CD40 band was detected in control cell lysate and HLE-untreated cell lysate, but not in HLE-treated cell lysate or supernatants from HLE-treated and untreated cells (Fig. 4B) . Furthermore, purified cell membranes of HGF were treated with HLE, and the cell membrane was analyzed by Western blotting using anti-CD40 polyclonal Ab. In untreated cell membranes, a strong 50-kDa CD40 band was detected, and the intensity of the CD40 band was reduced in a time-dependent manner. The CD40 band mostly disappeared at 60 min on HLE treatment. No fragments of CD40 were detected at any time point. Figure 4A 4B 4C , shows that reduction of CD40 by HLE resulted from direct proteolysis, but not from shedding or internalization following cell activation, and could be degraded into multiple CD40 fragments, which were not detected by polyclonal Ab for CD40.



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  		Figure 4. Cleavage of CD40 on fixed HGF and on HGF membrane by HLE treatment. (A) IFN-{gamma}-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).

 
Effect of HLE treatment on IL-8 and MCP-1 production from HGF triggered by CD40L
It has been reported that fibroblasts treated with IFN-{gamma} produce cytokines/chemokines in response to CD40L stimulation, and this production was up-regulated by IFN-{gamma} 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-{gamma}-treated HGF in a dose-dependent manner (Fig. 5A and B), but significant production was not detected from non-IFN-{gamma}-treated HGF (data not shown). Therefore, this series of experiments in Figure 5 was performed using IFN-{gamma}-treated HGF. Preincubation of IFN-{gamma}-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{alpha} 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{alpha} (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.



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  		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{alpha} (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).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we showed that HLE cleaved CD40 on HGF and resulted in the down-regulation of IL-8 and MCP-1 productions triggered by CD40L. This finding suggests that HLE has a feedback activity in inflammatory processes. Fibroblasts are not a homogeneous population, but can differ in morphology, phenotype, and function between and even within tissue types [2 ]. With regard to CD40, fibroblasts derived from various tissues such as dermis [5 ], lung [5 ], gingiva [5 , 12 ], periodontal ligament [8 ], or synovial membrane [6 ] express CD40 and are up-regulated by IFN-{gamma} 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-{alpha} [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.


   ACKNOWLEDGEMENTS
 
This work was supported in part by Grants-in-Aid for Scientific Research (12470469) and for Encouragement of Young Scientists (12771320 and 14771215) from the Japan Society for the Promotion of Science. We thank H. Takada and S. Sugawara for helpful discussions and T. Tsubahara, S. Kanaya, and G. Mayanagi for expert technical assistance in the preparation of HGF and myeloid lineage cells. We also thank D. Mrozek (Medical English Service, Kyoto, Japan) for reviewing the paper.

Received February 22, 2002; revised April 16, 2002; accepted April 18, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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