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(Journal of Leukocyte Biology. 2002;71:89-98.)
© 2002 by Society for Leukocyte Biology

Regulation of gelatinase B in human monocytic and endothelial cells by PECAM-1 ligation and its modulation by interferon-beta

Inge Nelissen, Isabelle Ronsse, Jo Van Damme and Ghislain Opdenakker

Rega Institute for Medical Research, Laboratory of Molecular Immunology, University of Leuven, B-3000 Leuven, Belgium

Correspondence: Prof. G. Opdenakker, Rega Institute for Medical Research, Laboratory of Molecular Immunology, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail: ghislain.opdenakker{at}rega.kuleuven.ac.be


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ABSTRACT
 
Platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) and gelatinase B are coexpressed at sites of inflammation, where an intense interaction occurs between leukocytes and endothelial cells. To investigate whether a functional link exists between PECAM-1 activation and gelatinase B production, the regulatory role of PECAM-1, IFN-{gamma}, IFN-ß, LPS, and PMA on the production of gelatinase B (MMP-9) was studied in vitro in normal human umbilical vein endothelial cells (HUVECs), human peripheral blood mononuclear cells (PBMCs), and in a human monocytic leukemia cell line. In THP-1 cells, progelatinase B levels were slightly up-regulated by immobilized PECAM-1-specific monoclonal antibody (mAb) and soluble recombinant PECAM-1 when compared with strong induction by LPS and PMA. IFN-ß inhibited the induced and basal gelatinase B production but had no modulating effect on the expression of PECAM-1. HUVECs mainly produced progelatinase A (proMMP-2). Treatment with LPS and triggering of the endothelial cells with PECAM-1 mAb or recombinant PECAM-1 had no effect on gelatinase A or B production, whereas PMA stimulated the production of progelatinase B. IFN-ß significantly up-regulated the expression of PECAM-1 in HUVECs but did not affect gelatinase secretion. Finally, in PBMCs, progelatinase B production was increased by soluble PECAM-1 mAb, recombinant PECAM-1, LPS, and PMA, whereas IFN-ß reduced gelatinase B secretion. IFN-ß did not alter PECAM-1 expression on PBMCs. Thus, PECAM-1 and gelatinase B are differently regulated in leukocytes and endothelial cells.

Key Words: multiple sclerosis • inflammation • cell-cell interaction • THP-1 • HUVEC • matrix metalloproteinase-9


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INTRODUCTION
 
Gelatinase B (MMP-9) is a complex matrix metalloproteinase in terms of multi-domain structure and levels of regulation of activity [1 ]. It plays a crucial role in leukocyte migration from the bone marrow into the circulation and from the circulation to inflamed tissues [2 ]. Gelatinase B expression has been associated with autoimmune diseases, e.g., multiple sclerosis (MS), by its presence in the tissues under attack and by its cleavage of substrate proteins into immunodominant epitopes [3 ]. Elevated expression by reactive astrocytes and macrophages is observed in demyelinating lesions in MS-affected brain, compared with normal brain tissue [4 , 5 ], and high levels of gelatinase B are detected in the cerebrospinal fluid and serum of MS patients [6 7 8 ]. In the bone marrow, gelatinase B enables the influx of leukocytes into the circulation leading to cytokine- or chemokine-induced leukocytosis [9 ]. In the periphery, gelatinase B is capable of disrupting the blood-brain barrier (BBB) by the destruction of extracellular matrix and myelin components [10 , 11 ]. Because the gelatinase B gene itself seems not to contribute to MS susceptibility [12 ], transactivation and gene regulation seem crucial in the disease. The understanding of leukocyte and endothelial cell (patho)physiology in the bone marrow and peripheral tissues, such as MS lesions, that leads to gelatinase B expression, may be helpful to define new ways to switch off the detrimental protease load.

Many types of soluble factors have been shown to influence the cellular production of gelatinase B: cytokines, chemokines such as interleukin (IL)-8, plant lectins, viral and bacterial products, and tumor promoters [13 14 15 16 17 18 ]. Although less well studied, cellular interactions through adhesion molecules have also been shown to induce gelatinase B [19 20 21 22 ].

One crucial cellular interaction in inflammation is between leukocytes and endothelial cells. In this interaction, the ligation of the platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a primary event. PECAM-1 is a highly abundant cell-surface glycoprotein capable of mediating homophilic and heterophilic adhesion. It is constitutively expressed and concentrated at the lateral borders between endothelial cells and expressed on the surfaces of neutrophils, monocytes, platelets, and some T-cell subsets [23 ]. In MS, PECAM-1 has been suggested to be a marker of disease activity [24 ], and increased levels of soluble PECAM-1 were found in sera of patients with active lesions, as diagnosed by magnetic resonance imaging (MRI), but not in those of patients without enhancing lesions [25 ]. In a rat model of experimental autoimmune encephalomyelitis (EAE), PECAM-1 expression is confined to endothelial cells of the BBB and is only mildly increased after induction of inflammation [26 ].

In view of the universal interactions between leukocytes and endothelial cells and the above-mentioned phenomenological studies on PECAM-1, gelatinase B, and MS, we investigated in vitro whether and how endothelial cells and monocytes as partners in adhesion may play a regulatory role in gelatinase B expression through homotypic PECAM-1 ligation. Also, because interferon-ß (IFN-ß) has been shown to modulate MMP-9 production in vitro in lymphocytes and monocytes [27 , 28 ] and because this cytokine has a beneficial influence on the course of relapsing-remitting MS, the possible modulating effect of IFN-ß on expression of PECAM-1 and gelatinase B in endothelial and monocytic cells was investigated. Similarly, the effect of the cytokine IFN-{gamma}, which is known to play a disease-promoting and proinflammatory role in MS [29 ], on PECAM-1 expression and gelatinase B secretion was examined.


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MATERIALS AND METHODS
 
Cell cultures
Human monocytic THP-1 cells were grown in stationary culture flasks in RPMI-1640 medium (BioWhittaker Europe, Verviers, Belgium), supplemented with sodium bicarbonate and 10% (v/v) fetal bovine serum (37°C, 5% CO2). During induction experiments with soluble inducers, the THP-1 cells were cultured in 24-well plates (1 ml/well; Nunc, Roskilde, Denmark) under serum-free conditions at 1 or 3 · 106 cells/ml. Cell numbers and viability were determined by counting in a Bürker hemacytometer after vital staining with trypan blue. Human mononuclear cells (MCs) were isolated from fresh heparinized peripheral blood (PB) from single donors (Red Cross Blood Transfusion Centre of Antwerp, Belgium). Leukocytes were fractionated by gradient centrifugation (400 g, 30 min) on Ficoll-sodium diatrizoate (Lymphoprep, Nycomed Pharma, Oslo, Norway), and the MC fraction (90% purity) was seeded in 24-well plates at 5 · 106 cells/ml/well in serum-free Eagle’s minimum essential medium (EMEM, BioWhittaker Europe) containing sodium bicarbonate and L-glutamine. Mononuclear cell cultures were enriched with monocytes by adherence of the latter cells to the bottom of the wells (2 h, 37°C, 5% CO2), followed by two washes with serum-free EMEM. After this adherence step, the cultures contained approximately 26% monocytes and 56% lymphocytes, as determined by flow cytometry after identification of the lymphocytes with CD3-staining. Inductions of peripheral blood mononuclear cells (PBMCs) were done in serum-free medium (500 µl/well) to which the inducers were added at various concentrations. All cell cultures were incubated at 37°C (5% CO2) for various time intervals.

In solid-phase induction experiments, 96-well plates (Maxisorp, Nunc) was coated with the inducer at various concentrations for 3 days at 4°C. After three washes with phosphate-buffered saline (PBS; without Ca2+ and Mg2+), followed by a blocking step with 2.5% low-endotoxin bovine serum albumin (Sigma Chemical Co., St. Louis, MO) and three additional wash steps, THP-1 or PBMC cultures (200 µl/well) were added to the precoated plates in serum-free medium.

Human umbilical vein endothelial cells (HUVECs, pooled from several donors, Clonetics, BioWhittaker Europe) were cultured in endothelial cell growth medium (EGM®-2 Bulletkit, Clonetics, BioWhittaker Europe) supplemented with 2% fetal bovine serum. The HUVECs were used in subsequent passages and were grown to subconfluence in 24-well plates at 37°C, 5% CO2 before stimulation with various inducers. In coculture experiments, THP-1 cells or trypsinized PBMCs were seeded onto the HUVEC monolayers.

Antibodies and reagents
The following antibodies were used: purified mouse anti-human monoclonal antibody (mAb) against CD3 (clone UCHT1, 0.5 mg/ml), purified mouse anti-human mAb against PECAM-1 (clone WM-59, 0.5 mg/ml), and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin (Ig)-specific polyclonal antibody. All mAbs were of the IgG1 isotype and purchased from Becton Dickinson PharMingen (Erembodegem, Belgium). Affinity-purified mouse IgG1,{kappa} monoclonal Ig isotype standard from mouse ascites (clone MOPC-21, 0.5 mg/ml) was used as an irrelevant control antibody (Becton Dickinson PharMingen). Human recombinant CD31 (further denoted as hrPECAM-1; expressed by Chinese hamster ovary cells; 1 mg/ml) was purchased from R&D Systems (Oxon, UK). Human recombinant IFN-{gamma} (Bioferon, Laupheim, Germany; 50,000 U/ml in serum-free EMEM) was purchased, and human recombinant IFN-ß [30 ] (30,000 U/ml in PBS) was produced and purified at the Rega Institute (Leuven, Belgium). Phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co.; 100 µg/ml) and bacterial lipopolysaccharide (LPS; Difco Laboratories, Detroit, MI; 1 mg/ml) were diluted in PBS. Stromelysin-1 (MMP-3; Biogenesis, Poole, UK) was activated with 4-aminophenylmercuric acetate (APMA) as described [31 ].

A constant concern of induction experiments with recombinant products and mAbs is endotoxin contamination. To exclude that the observed gelatinase B induction levels were a result of endotoxin contamination of the preparations, endotoxin levels of the commercial preparations were determined by the Limulus amebocyte lysate test (QCL-1000, BioWhittaker Europe). Endotoxin contents in the commercial PECAM-1 mAb and hrPECAM-1 preparations were 930 and 300 pg/ml endotoxin, respectively, whereas the endotoxin content in the IgG1,{kappa} isotype standard preparation was below the detectable level (<10 pg/ml). These concentrations did not induce gelatinase B expression above background levels, as confirmed by zymography analysis of supernatants from THP-1 cells, treated with various concentrations of endotoxin.

Flow cytometry
For fluorescent-activated cell sorter (FACS) analysis, cell monolayers (PBMCs and HUVECs) were detached from 24-well plates by washing with N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES)-buffered saline solution containing 30 mM HEPES (Clonetics, BioWhittaker Europe), followed by incubation in 0.025% trypsin solution and 0.01% ethylenediaminetetraacetate (EDTA) in HEPES (Clonetics, BioWhittaker Europe) and collection with trypsin-neutralizing solution (Clonetics, BioWhittaker Europe). Suspension cells (THP-1) were collected by pipetting up and down from the culture wells. The cells were then incubated for 30 min on ice with saturating concentrations (1 µg/106 cells) of a primary mouse anti-human mAb. After washing with PBS, the cells were incubated for 30 min with a 1:200 dilution of FITC-labeled goat anti-mouse Ig-specific polyclonal antibody (0.5 mg/ml) in the dark and analyzed on a FACStar flow cytometer (Becton Dickinson). Finally, cells were resuspended in PBS containing 0.5% (v/v) formaldehyde before flow cytometry analysis. Controls consisted of cells stained with commercial isotype-matched irrelevant murine monoclonal Ig. Cells were gated using forward- versus side-scatter to exclude dead cells and debris. For assessment of antigen expression, gated numbers of THP-1 cells, HUVECs, and PB monocytes were 20,000, 15,000, and 15,000, respectively. Data collection and analysis were done by using the CELLQuest software program (Becton Dickinson).

mRNA Isolation and Northern blot hybridization
Total cellular RNA was isolated by the guanidium thiocyanate method (RNeasy® Mini kit, Qiagen, Westburg, Leusden, the Netherlands) from resting or PECAM-1 mAb-treated THP-1 cells. RNA samples were denatured, separated on a 1.5% agarose gel (10 µg RNA/lane) in the presence of ethidium bromide, and blotted onto a nylon membrane (Hybond-XL, Amersham Pharmacia Biotech, Uppsala, Sweden) by capillary transfer for 3.5 h using the NorthernMaxTM-Gly kit (Ambion, Austin, Texas). RNA was hybridized to cDNA probes that were radioactively labeled with [{alpha}32P]dCTP (Amersham Pharmacia Biotech) by a random priming procedure using the Klenow DNA polymerase I (MegaprimeTM DNA labeling kit, RPN 1607, Amersham Pharmacia Biotech). Hybridizations were performed for at least 1 h at 68°C in ExpressHybTM hybridization solution (Clontech Laboratories, Palo Alto, CA). The membrane was washed under high-stringency conditions, and the signals were revealed by autoradiography on a Kodak BioMax film. The cDNA encoding human gelatinase B was kindly obtained from Dr. G. Goldberg [32 ], whereas the cDNA encoding human elongation factor-1{alpha} (pAB48K) was used to verify the intactness of the mRNA and the processing of the samples [33 ].

Zymography
Cell cultures were normalized to cell number prior to stimulation with various inducers. Conditioned cell supernatants were supplemented with loading buffer [125 mM Tris-HCl, pH 6.8, 0.1% (w/v) bromophenol blue, 4% (w/v) sodium dodecyl sulfate (SDS), 8.5% (w/v) sucrose] and loaded onto 1% (w/v) SDS/7.5% (w/v) polyacrylamide gels containing 0.1% (w/v) gelatin (Sigma Chemical Co.). Equal supernatant volumes were loaded within a gel. Electrophoresis was performed overnight at 4°C in Tris-glycine buffer containing 0.1% (w/v) SDS. For molecular weight standardization, the molecular weight standard mixture [SDS-polyacrylamide gel electrophoresis (PAGE) molecular weight standards, BioRad Laboratories, Nazareth, Belgium] consisted of myosin (200,000 Da), ß-galactosidase (116,250 Da), phosphorylase b (97,400 Da), serum albumin (66,200 Da), and ovalbumin (45,000). Subsequently, gels were incubated twice for 20 min in washing buffer (50 mM Tris-HCl, pH 7.5, 10 mM CaCl2, 0.02% (w/v) NaN3) containing 2.5% (v/v) Triton X-100 (Sigma Chemical Co.) to remove SDS. To detect gelatinolytic activity, the gel was incubated overnight at 37°C in the washing buffer containing 1% (v/v) Triton X-100 (reaction buffer). Enzymatic activity was revealed by staining the gel with 0.25% (w/v) Coomassie Brilliant Blue R-250 (Sigma Chemical Co.) in 45% (v/v) methanol/10% (v/v) glacial acetic acid and destaining in 30% (v/v) methanol/10% (v/v) acetic acid. Gelatinase activity was detected as unstained bands on a blue stained background. Scanning densitometry was used to quantify the gelatinolytic activity of the samples [18 ].


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RESULTS
 
Modulation of PECAM-1 expression in human promonocytic THP-1 cells, HUVECs, and PBMCs after stimulation with IFN-ß and IFN-{gamma}
IFN-ß and IFN-{gamma} do not modulate PECAM-1 expression of THP-1 cells
Because PECAM-1 expression is present in inflammatory lesions, assists in leukocyte endothelial cell interaction [34 ], and may help as costimulator in other cellular interactions that lead to gelatinase B induction, it was first studied whether the anti-inflammatory cytokine IFN-ß and the pro-inflammatory cytokine IFN-{gamma} had an effect on PECAM-1 expression. THP-1 cells were seeded and supplemented with IFN-ß or IFN-{gamma} (1000 U/ml) and with LPS (10 µg/ml) or PMA (10 ng/ml) to evaluate the inducibility of PECAM-1. Cells and supernatants were harvested after 24 and 48 h. Basal expression levels of PECAM-1 were confirmed to be high for nonstimulated THP-1 cells, as measured by flow cytometry (Fig. 1 A ). Treatment with IFN-{gamma}, IFN-ß, and LPS did not affect PECAM-1 expression after 24 h, and a small but insignificant increase in the mean fluorescence intensity (MFI) was measured after 48 h incubation. Exposure of the cells to PMA slightly up-regulated the expression of PECAM-1 from 24 h onward.



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  		Figure 1. Flow cytometric analysis of PECAM-1 expression on THP-1 cells (A), HUVECs (B), and peripheral blood monocytes (C). Cells were treated for 24 or 48 h with IFN-{gamma} (100 U/ml for monocytes; 1000 U/ml for THP-1 and HUVECs), IFN-ß (100 U/ml for monocytes; 1000 U/ml for THP-1 and HUVECs), and LPS (10 µg/ml) and PMA (10 ng/ml; solid line) or were treated with an equivalent volume of solvent (dotted line) before staining with anti-PECAM-1 (clone WM-59). Controls consisted of untreated cells that were incubated with an isotype-matched irrelevant mAb (clone MOPC-21; dashed line). The dot plots show the side (SSC)- versus forward (FSC)-scatter for control cells, and R indicates the gate, containing the cells that are analyzed in the histograms. The numbers in the histograms indicate the MFIs. The control values of reactions with the negative background antibody or of solvent-treated cells are indicated in italics, whereas the bold numbers are from stimulated cells. The presented data are representative of two (monocytes) or three (THP-1 and HUVECs) independent experiments.

IFN-ß and IFN-{gamma} up-regulate PECAM-1 expression of HUVECs
To investigate modulating effects on PECAM-1 expression by HUVECs, subconfluent cultures were incubated for 24 and 48 h with the same inducers as the THP-1 cells. As measured by flow cytometry, treatment of HUVECs with IFN-ß (1000 U/ml) resulted in a significant increase of the MFI with respect to control cultures supplemented with PBS (MFI=664 vs. 368 after 48 h; Fig. 1B ). Similar observations were made in the presence of PMA after 48 h incubation (MFI=636 vs. 368) and also with 1000 U/ml IFN-{gamma} (MFI=532 vs. 401 for EMEM-treated controls), but the latter effect was less pronounced. All three effects were time-dependent. When the cell cultures were induced with LPS, initially the MFI was slightly enhanced but reduced again after 48 h compared with control.

IFN-ß and IFN-{gamma} do not alter PECAM-1 expression of normal monocytes
PBMCs were prepared as described in Materials and Methods and treated with several inducers. Monocytes were distinguished from the lymphocytes by CD3-staining of the total cell population. By flow cytometry, 60,000 positively staining MCs were gated, so that approximately 15,600 monocytes (26%) could be studied separately. Basal expression of PECAM-1 by normal monocytes was unaffected after treatment with IFN-ß (100 U/ml) or IFN-{gamma} (100 U/ml) for 24 h (Fig. 1C) . Conversely, LPS and PMA strongly reduced the MFI from 24 h onward, compared with untreated cells. In fact, the total numbers of primary monocytes were highly decreased after treatment with LPS and PMA compared with control cultures (respectively, 39% and 19% of the monocyte number in control cultures after 24 h). The different inducers did not alter the expression of PECAM-1 by PB lymphocytes or the number of lymphocytes in the culture (unpublished results).

THP-1 cells, HUVECs, and PBMCs produce latent progelatinases after stimulation
Preliminary experiments were carried out to determine whether the cell types used in this study produce the latent progelatinase B, the activated enzyme form, or both. Because various processed forms of macrophage and neutrophil 92-kDa gelatinase B are obtained with stromelysin-1 and APMA [35 ], the latter two substances were used to treat conditioned culture fluids of THP-1 cells, HUVECs, and PBMCs.

Promonocytic THP-1 cells produce a 96-kDa gelatinase B after stimulation with PMA, IL-1ß, LPS, and the lectin concanavalin A (Con A) [36 ]. To investigate whether the THP-1 gelatinase B constituted the proenzyme form, THP-1 cells were stimulated with 10 µg/ml LPS for 48 h. The presence of (pro)gelatinases A and B in the culture medium was confirmed by gelatin zymography. Subsequently, supernatants were incubated at 37°C with (APMA-activated) stromelysin-1 in an estimated molar ratio stromelysin-1:gelatinase B of 20:1 or with APMA in a final concentration of 10 mM for different time intervals and were analyzed. Already within 15 min after treatment with stromelysin-1, lower molecular weight forms of the processed gelatinase B with gelatinolytic activity appeared. Incubation with APMA did not efficiently convert the progelatinase B to a lower molecular weight form but rather degraded the proenzyme. Indeed, the levels of progelatinase B diminished without the concomitant increase of activated enzyme. Similar observations were made for the processing of the proenzyme form of gelatinase A. At least two activated forms of progelatinase A were observed after treatment with stromelysin-1, whereas degradation of progelatinase A was observed with APMA.

In HUVECs it was demonstrated previously that the production of a 92-kDa gelatinase B is stimulated by PMA [37 ]. Therefore, subconfluent HUVEC cultures were supplemented with 10 ng/ml PMA and incubated for 48 h. Conditioned media were subsequently treated with stromelysin-1 (molar ratio progelatinase:stromelysin, 1:20) or with APMA (10 mM) for different time intervals and analyzed by zymography. Without the addition of stromelysin-1 or APMA, weak production of progelatinase B was observed, whereas progelatinase A and some processed forms of the latter enzyme were abundantly secreted in the culture medium. Incubation with stromelysin-1 weakly altered the processing status of progelatinases A and B, as was observed by the appearance of small amounts of lower molecular-weight products, whereas treatment with APMA resulted in a further conversion of the progelatinase A but not of progelatinase B.

PBMC cultures were treated with PMA (10 ng/ml) for 48 h and subsequently treated with stromelysin-1 or APMA as described before. The pattern of progelatinase B after activation resembled that of processed THP-1 progelatinase B, although processing by stromelysin-1 and degradation by APMA were more complete. Basal progelatinase A production was barely detectable in culture fluids of primary blood cells but became visible on the gel after treatment of the cells with stromelysin-1 and showed similar processing products as the THP-1 progelatinase A (data available on request).

Effect of specific ligation of PECAM-1 on gelatinase B production by THP-1 cells, HUVECs, and PBMCs
Gelatinase B production is up-regulated in THP-1 cells by specific ligation of PECAM-1
PECAM-1 interacts with itself in a homotypic way [23 ]. To study cellular interaction by PECAM-1 ligation and the effect on gelatinase B production, THP-1 cells were seeded at 1.106 cells/ml in multi-well plates and stimulated with soluble hrPECAM-1 (2 and 20 µg/ml) or PECAM-1 mAb (2 and 20 µg/ml). Supernatants were harvested after 24, 48, and 72 h and analyzed for gelatinolytic activity by substrate zymography (Fig. 2 A ). Gelatinases A and B were identified on the gels as proenzyme forms. Gelatinase B levels were not increased in cells, stimulated with soluble PECAM-1 mAb (20 µg/ml; lane 5), whereas stimulation of the cells with soluble hrPECAM-1 (20 µg/ml; lane 10) and LPS (10 µg/ml; lane 11) or PMA (10 ng/ml; lane 15) as positive controls for inducibility of the cells up-regulated gelatinase B production to levels that were significantly higher than background levels (lane 19).



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  		Figure 2. Modulation of gelatinase production in THP-1 cells by PECAM-1 mAb, hrPECAM-1, LPS, or PMA and effect of IFN-ß on gelatinase production after stimulation. (A) THP-1 cultures (1.106 cells/ml) were supplemented with soluble PECAM-1 mAb (20 µg/ml; lane 5), hrPECAM-1 (20 µg/ml; lane 10), LPS (10 µg/ml; lanes 11–14), or PMA (10 ng/ml; lanes 15–18) or were left untreated (Control; lanes 19–22). Furthermore, THP-1 cells were seeded in 96-well plates that were precoated with PECAM-1 mAb (20 µg/ml; lanes 1–4) or hrPECAM-1 (20 µg/ml; lanes 6–9). Cultures were co-treated with IFN-ß (0, 10, 100, or 1000 U/ml) and incubated for 48 h, and gelatinases A (GA) and B (GB) production levels were assessed in the conditioned media (20 µl/lane) by zymography. (B) THP-1 cells (3.106 cells/ml) were added to 96-well plates, precoated with dilution series of PECAM-1 mAb (0.7–60 µg/ml) and IgG1,{kappa} isotype standard (0.7–60 µg/ml). Conditioned supernatants were harvested after 24 h and analyzed by gelatinase zymography. Nontreated cells (denoted as C) were used as controls. (C) THP-1 cells (1.106 cells/ml) were cultured in 96-well plates that were precoated with PECAM-1 mAb (16.7 µg/ml), stimulated with soluble LPS (10 µg/ml) or PMA (10 ng/ml), or were left untreated (Control). Cultures were co-treated with IFN-ß (0, 10, 100, or 1000 U/ml) and incubated for 48 h. Supernatants were analyzed by zymography.

It remained possible that the trigger for PECAM-1 mAb-mediated signal transduction necessitates multi-valent activation. Therefore, solid-phase activation of the cells, which also leads to multi-valent synapse-like interactions, was performed to mimic cell-cell interaction. After exposure to immobilized PECAM-1 mAb (20 µg/ml; lane 1), THP-1 cells released increased levels of gelatinase B in the culture media, whereas exposure to immobilized hrPECAM-1 (20 µg/ml; lane 6) did not alter gelatinase B production. After triggering with immobilized anti-PECAM-1 (0.7–60 µg/ml), gelatinase B secretion presented a dose- and time-dependent increase (Fig. 2B) . An immobilized irrelevant IgG1,{kappa} control antibody, included in three independent induction experiments, induced gelatinase B to levels that were significantly lower than those with the PECAM-1 mAb.

The effect of the anti-inflammatory cytokine IFN-ß on gelatinase B production in THP-1 cells, induced with immobilized PECAM-1 mAb and hrPECAM-1 and with soluble LPS and PMA, was studied after 24, 48, and 72 h (Fig. 2A and 2C) . IFN-ß (10, 100, and 1000 U/ml) was added simultaneously with the cells to the precoated wells. The up-regulated gelatinase B production could not be inhibited by IFN-ß at doses below 1000 U/ml. However, in various independent experiments, a 1000 U/ml dose of IFN-ß reproducibly down-modulated the gelatinase B levels, induced by the different agonists. IFN-ß did not alter gelatinase B secretion levels of THP-1 cells that were not pretreated.

Immobilized PECAM-1 mAb slightly up-regulates THP-1 gelatinase B mRNA
The expression of gelatinase B mRNA in THP-1 cells after stimulation with immobilized PECAM-1 mAb (20, 10, 5, and 2.5 µg/ml) and soluble LPS (10 µg/ml) was studied by Northern blot analysis. Up-regulation of gelatinase B transcription by PECAM-1 mAb was detected, compared with negative controls, but was rather weak compared with up-regulation by LPS (Fig. 3 ), as corroborated at the protein level (Fig. 2) . Sample processing and loading were controlled by reprobing the filter with a cDNA probe of the human elongation factor-1{alpha}.



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  		Figure 3. Effect of immobilized PECAM-1 mAb on GB mRNA levels in THP-1 cells. PECAM-1 mAb (20, 10, 5, and 2.5 µg/ml) was immobilized onto the surface of a microtiter plate, and THP-1 cells (1.106 cells/ml) were seeded in the precoated wells. As positive controls, THP-1 cells were treated with LPS (10 µg/ml) or PMA (10 ng/ml). Control A and Control B represent negative controls without and with addition of bovine serum albumin in the blocking step of the immobilization procedure. After 16 h of incubation, total RNAs were extracted from the cells, and gelatinase B mRNA was visualized by Northern blot analysis. After stripping of the gelatinase B probe, the filter was reprobed with cDNA encoding human elongation factor-1{alpha} (EF-1{alpha}).

Gelatinase B production is not modulated in HUVECs by specific ligation of PECAM-1
The possible implication of PECAM-1 in the induction of gelatinase B in HUVECs was investigated in vitro by adding PECAM-1 mAb or hrPECAM-1 to the culture medium of HUVECs, seeded in 24-well plates, and grown to subconfluence. Production of (pro)gelatinase B was low in nontreated cells, whereas high basal levels of (pro)gelatinase A were observed by zymographic analysis (Fig. 4 , Control lanes). Depending on the activation status of the cells, both metalloproteinases showed activation to lower molecular-weight forms. Activated forms of gelatinase A became more clear as the cells were incubated for a longer time interval (48 and 72 h). Addition of PECAM-1 mAb or hrPECAM-1 (2 and 20 µg/ml) did not increase expression nor did it alter the activation status of both gelatinases, compared with nonstimulated HUVECs at the different time points observed (unpublished results). When HUVEC cultures were exposed to LPS (10 µg/ml), no further stimulation or processing of gelatinase A or B was detected when compared with untreated cultures. In contrast, treatment of the cells with PMA (10 ng/ml) resulted in a weak up-regulation of gelatinase B, whereas the production level of gelatinase A remained unaffected. However, gelatinase A showed more processing after PMA treatment from 24 h incubation onward as compared with control cultures (Fig. 4) . When HUVEC cultures were exposed to immobilized PECAM-1 mAb or hrPECAM-1 (20 µg/ml), basal gelatinase levels and activation of the proenzymes were not affected. When these cultures were co-treated with IFN-ß (10, 100, and 1000 U/ml), no effect of IFN-ß on gelatinase production was observed (unpublished results).



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  		Figure 4. Effect of LPS and PMA on GA and GB production in HUVECs. Subconfluent HUVEC cultures were supplemented with LPS (10 µg/ml) or PMA (10 ng/ml) and incubated for 24, 48, or 72 h. Supernatants were collected, and 20 µl was analyzed by zymography. Controls consisted of untreated cultures. A molecular weight standard (lane M), indicated in kDa, was used for size calibration.

Gelatinase B production is up-regulated in PBMCs by specific ligation of PECAM-1
Cultures of PBMCs were treated with the soluble inducers PECAM-1 mAb (20 µg/ml), recombinant PECAM-1 (20 µg/ml), LPS (10 µg/ml), and PMA (10 ng/ml). Equivalent aliquots of conditioned PBMC supernatants were used to qualitatively evaluate the presence of gelatinases after 24 and 48 h incubation by zymography analysis. Compared with control cultures and as quantified from three independent experiments, gelatinase B production was increased after 24 h in cultures treated with PMA (1.6-fold), hrPECAM-1 (1.5-fold), LPS (1.4-fold), and PECAM-1 mAb (1.2-fold; Fig. 5 ). After 48 h incubation, gelatinase B levels were still slightly up-regulated compared with controls but to a lesser extent. When these cultures were co-treated with IFN-ß (100 and 1000 U/ml), a dose of 1000 U/ml clearly inhibited the induced gelatinase levels. Treatment of control PBMC cultures with IFN-ß (1000 U/ml) also down-modulated background gelatinase B levels (2.5-fold after 24 h and 1.4-fold after 48 h), whereas 100 U/ml IFN-ß had no effect. PBMC gelatinase A was not detected on the gelatin gels.



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  		Figure 5. Modulation of GB production in PBMCs by PECAM-1 mAb, hrPECAM-1, LPS, and PMA and antagonization of stimulated gelatinase production by IFN-ß. PBMC cultures were supplemented with soluble PECAM-1 mAb (20 µg/ml), hrPECAM-1 (20 µg/ml), LPS (10 µg/ml), or PMA (10 ng/ml) or left untreated (Control lanes). All cultures were co-treated with IFN-ß (0, 100, or 1000 U/ml). After incubating the cultures for 24 h, gelatinase production was assessed in the conditioned media (10 µl/lane) by zymography.

Production of gelatinases in cocultures of HUVECs with THP-1 or PBMCs
Gelatinase secretion by endothelial cells and leukocytes was observed when both cell types were allowed to interact with each other. In a first experiment, HUVECs were cocultured with THP-1 cells (ratio 4:1) or with PBMCs (ratio 1:1.5), and conditioned media were analyzed by zymography after 24 and 48 h (Fig. 6 A ). Compared with single-cell control cultures of HUVECs (lanes 6 and 13) and THP-1 cells (lanes 7 and 14), gelatinases A and B showed no altered levels in the coculture of both cell types (lanes 5 and 12). Gelatinase levels, produced by the individual cell types, were superposed on each other. The coculture of HUVECs and PBMCs (lanes 2 and 9) also resulted in additive gelatinase production, and the levels were similar compared with those of HUVEC (lanes 3 and 10) and PBMC cultures (lanes 4 and 11) alone. To determine the effect of PECAM-1 ligation on gelatinase secretion by the latter coculture, HUVEC cultures were treated with PECAM-1 mAb (60 µg/ml) during 2 h before the PBMC cultures were added (lanes 1 and 8). No increase in gelatinase B production was observed by PECAM-1 ligation compared with control cocultures.



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  		Figure 6. Production of GA and GB in cocultures of HUVECs with PBMCs or THP-1 cells. (A) PBMCs or THP-1 cells were cocultured with HUVECs (ratios 1.5:1 and 1:4, respectively) after the HUVECs had grown to subconfluence in 24-well plates (350,000 cells per well). Subconfluent HUVEC cultures were previously left untreated (lanes 2–7 and lanes 9–14) or pretreated with 60 µg/ml PECAM-1 mAb for 2 h (lanes 1 and 8). After 24 and 48 h, conditioned coculture media were collected and analyzed by zymography. HUVEC-PBMC gelatinase production is presented in lanes 1, 2, 8, and 9, whereas HUVEC-THP-1 gelatinase is shown in lanes 5 and 12. Controls consisted of HUVECs, PBMCs, or THP-1 cells cultured separately at the same cell density compared with the density in the cocultures. (B) PBMCs or THP-1 cells were cocultured with subconfluent HUVEC monolayers (150,000 cells per well; ratios 1:1) after the PBMC or THP-1 cultures were previously left untreated (lanes 1 and 8 and lanes 3 and 10, respectively) or pretreated with 60 µg/ml PECAM-1 mAb for 1 h (lanes 2 and 9 and lanes 4 and 11, respectively). Gelatinase production was analyzed in conditioned coculture media after 24 and 48 h by zymography. Pure HUVEC, PBMC, or THP-1 cell cultures were used as controls and contained equivalent cell numbers as these in the cocultures.

In a second type of experiment, PBMC and THP-1 cultures were treated with 60 µg/ml PECAM-1 mAb, 1 h prior to coculturing with HUVECs, because ligation of PECAM-1 on THP-1 cells and PBMCs increased gelatinase B production (Fig. 6B) . Compared with single-cell cultures (lanes 5–7 and lanes 12–14), coculturing of HUVECs with untreated PBMCs (lanes 1 and 8) or THP-1 cells (lanes 3 and 10) resulted in additive induction levels of gelatinases A and B, but no synergistic effect was observed. PECAM-1 ligation of PBMCs slightly decreased gelatinase B levels in the cocultures with HUVECs (lanes 2 and 9), whereas no effect of PECAM-1 ligation of THP-1 cells on gelatinase production was observed in coculture with HUVECs (lanes 4 and 11).


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DISCUSSION
 
The present study was intended to define whether and how PECAM-1 and gelatinase B may play a functional role in leukocyte and endothelial cell biology. The expression of gelatinase B has become a hallmark of inflammation of the central nervous system, with MS as the prototype of autoimmune process [3 , 8 ]. By using gelatinase B as read-out and IFN-ß as a modulator, we were able to compare PECAM-1 as trigger and effector under the influence of soluble immunomodulators. The latter was done on in vitro cultures of myelomonocytic cells, PBMCs, and endothelial cells.

First, the modulating effect of different inducers on PECAM-1 expression was assessed by flow cytometry. In THP-1 cells, PECAM-1 expression was not significantly altered by IFN-ß, IFN-{gamma}, or LPS, but PMA slightly up-regulated PECAM-1. The latter observation is in accordance with the up-regulation of PECAM-1, which was previously shown in the leukemia cell lines U937 (promonocytic) and HL-60 (myelomonocytic) during PMA-induced differentiation [38 39 40 ]. In endothelial cells, PECAM-1 expression was enhanced by IFN-{gamma} and PMA and to a higher extent by IFN-ß. Stimulation with LPS reduced PECAM-1 levels when compared with control levels. In peripheral blood monocytes, PECAM-1 expression was not altered after 24 h incubation with IFN-{gamma} and IFN-ß, whereas the PECAM-1 expression, together with the number of viable cells, decreased remarkably after incubation with LPS and PMA.

Several studies support the observed promoting role of IFN-{gamma} on PECAM-1 expression. In HUVECs, it was found that the combination of IFN-{gamma} plus tumor necrosis factor {alpha} inhibits PECAM-1 synthesis, but this phenomenon was not found with each cytokine alone [41 ]. Furthermore, IFN-{gamma} contributes to the Herpes simplex virus type 1-induced corneal inflammation by facilitating polymorphonuclear neutrophil infiltration, which appears to be accomplished through up-regulation of PECAM-1 expression on the vascular endothelium [42 ]. In contrast, another study demonstrates a significant decrease of the percentage of PECAM-1-positive HUVECs in the presence of IFN-{gamma} alone or when combined with IL-10 [43 ].

In a next step, we tried to define the functional implication of PECAM-1 expression and ligation on gelatinase B production with the use of promonocytic, mononuclear, and endothelial cell cultures. Gelatinases A and B were mainly produced in the proform that was activatable by stromelysin-1. The organomercurial APMA was also an activator for progelatinase A in HUVECs, but in THP-1 cells and PBMCs, progelatinases A and B were degraded in the presence of 10 mM APMA. In contrast to soluble mediators, such as LPS or PMA, that result in a strong up-regulation of gelatinase B in THP-1 cells [36 ], homotypic ligation of PECAM-1 resulted in only weak induction of gelatinase B. The triggering with a PECAM-1-specific mAb was only effective after multi-meric (solid-phase) presentation. The latter up-regulation of gelatinase B expression was corroborated at the mRNA level. The induction of gelatinase B by LPS and PMA, as well as by PECAM-1 ligation, was antagonized by treatment of THP-1 cells with IFN-ß. In HUVECs, considerable levels of gelatinase A were constitutively expressed, whereas gelatinase B production was rather low. Ligation of PECAM-1 or exposure to LPS did not affect constitutive gelatinase levels in HUVECs. In contrast, PMA stimulated gelatinase B production and gelatinase A processing, as described earlier [37 , 44 ]. In PBMCs, gelatinase B production was slightly elevated by the soluble agonists LPS, PMA, and recombinant PECAM-1 and to a lesser extent, by the ligation with PECAM-1 mAb. It should be noticed that PBMCs had higher background levels of gelatinase B than THP-1 cells, which implies that induction levels were lower. Nevertheless, the demonstrated effects were reproducible (at least three experiments). Co-treatment of the PBMC cultures with 1000 U/ml IFN-ß inhibited the up-regulated enzyme levels.

IFN-ß is currently one of the treatments of MS and has been shown to down-modulate the expression of gelatinase B by lymphocytes after stimulation with IL-2 [28 ], and by PBMCs stimulated with MCP-1 or cocultured with activated human brain microvascular endothelial cells in vitro [45 , 46 ] and also in vivo during treatment of patients [47 , 48 ]. Our study extends these observations and shows that IFN-ß has a broad cell spectrum in down-modulating gelatinase production. Indeed, IFN-ß not only represses the gelatinase levels that are induced by soluble mediators but also the basal gelatinase levels and gelatinase B induced by PECAM-1 ligation. Therefore, the present study on gelatinase regulation by PECAM-1 has to be compared with other regulatory studies by cell adhesion molecules and soluble mediators. In contrast to the higher gelatinase B levels, induced by intercellular adhesion molecule-1 and lymphocyte function-associated antigen-1 ligation [21 ] or by CD40 ligation [49 ], the homotypic ligation of PECAM-1 results in a rather weak gelatinase production. Because PECAM-1 expression is constitutive on endothelial cells and leukocytes, the PECAM-1-mediated up-regulation of gelatinase B is suggested to occur mainly in the perivascular cuffs. IFN-ß may play a double protective role. By inducing PECAM-1 up-regulation, it may enhance settlement and residency of leukocytes in the perivasular cuffs (and decrease invasion into the central nervous system parenchyma). By decreasing gelatinase B production, the leukocyte infiltration and demyelination will be dampened. Indeed, homotypic ligation of PECAM-1 of leukocytes or leukemic cells with PECAM-1 from endothelial cells resulted only in an additive release of gelatinases A and B.

In conclusion, although there is circumstantial evidence for a role of PECAM-1 in inflammatory diseases, such as MS pathology [25 ], recent genetic studies [50 , 51 ], as well as the current functional data, favor the idea of an indirect role of PECAM-1. From the effects of IFN-ß on MS disease (beneficial) and on gelatinase B (down-modulation) and PECAM-1 (up-regulation), it is suggested that PECAM-1 expression may be associated with beneficial effects. The functional dissection of the complex interactions of cells, inducers, and effector molecules is not only crucial for the understanding of the mechanisms underlying diseases but also complements the current phenomenological studies and needs to be considered in the development of novel therapies.


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ACKNOWLEDGEMENTS
 
This work was supported by Fortis Insurances Belgium (FB), the Fund for Scientific Research (FWO-Vlaanderen), the Geconcerteerde Onderzoeksacties (GOA), and the Charcot Foundation, Belgium. I. N. is a research fellow of the Multiple Sclerosis Research Foundation (WOMS, 1998–2002). The authors appreciate the excellent technical assistance of Jean-Pierre Lenaerts for cell culture, Tania Mitera for flow cytometry, and Erik Martens for the induction experiments.

Received March 4, 2001; revised August 18, 2001; accepted August 20, 2001.


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