JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 21:495–501 (2001)Mary Ann Liebert, Inc.

Human Monocyte-Derived Dendritic Cells Produce BioactiveGelatinase B: Inhibition by IFN-b

EMMANUEL J. BARTHOLOMÉ,1 ILSE VAN AELST,4 ELS KOYEN,4 ROBERT KISS,3

FABIENNE WILLEMS,1,2 MICHEL GOLDMAN,1 and GHISLAIN OPDENAKKER4

ABSTRACT

We studied the secretion of gelatinase B by dendritic cells (DC) generated by culturing human peripheralblood monocytes in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4).First, we found the intracellular expression of gelatinase B on sections of fixed DC pellets. Zymography anal-ysis of the supernatants of DC cultured for 72 h demonstrated the presence of gelatinase B. To determine ifDC produce net enzymatic activity, bioactive gelatinase, a novel sensitive fluorescent-activated substrate con-version (FASC) assay was used to complement the zymography data. Culture media of unstimulated DC dem-onstrated reproducible net gelatinolytic activity. Tumor necrosis factor-a (TNF-a) IL-1b but not lipopolysac-charide (LPS) stimulation caused a significant increase in gelatinase B production in zymography analysis.Both types of stimulation failed to increase net gelatinase activity in FASC assay. Interestingly, interferon-b(IFN-b) significantly diminished both the total zymolytic production and the net bioactive gelatinase producedby DC in a dose-dependent manner. We conclude that human monocyte-derived DC secrete bioactive gelati-nase B and that IFN-b inhibits this production.

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INTRODUCTION

THE PIVOTAL ROLE OF DENDRITIC CELLS (DC) in the inductionof immune responses is now well established (reviewed in

ref. 1). Located in most tissues, immature DC actively captureand process antigens. In response to microbial products or in-flammatory cytokines, DC mature and migrate to lymphoid or-gans, where they activate antigen-specific T cells. However, lit-tle is known about the mechanisms involved in DC migrationthrough tissues and endothelial barriers.

Matrix metalloproteinases (MMP) have been identified askey enzymes for the degradation of extracellular matrices.(2)

These enzymes are thus considered to contribute as physiologicmediators in cell migration. Unlike gelatinase A (MMP-2),which is constitutively produced in various cell types, gelati-nase B (MMP-9) production is regulated by different stimuli.(3)

Increased levels of gelatinase B in body fluids have been as-sociated with a number of diseases, such as rheumatoid arthri-tis,(4) multiple sclerosis (MS),(5) and cancer cell invasion.(6)

Similarly, inhibition of MMP has been found to be beneficialin animal models of, for instance, allergic asthma(7) and MS.(8)

We have been studying the role of the MMP-9, gelatinaseB, in autoimmune diseases, particularly in MS.(3,5) In this dis-ease, it is well established that a type I interferon (IFN), IFN-b, has disease-limiting effects.(9,10) The mechanisms of theseeffects may be multiple. Several studies have documented thatIFN-b downmodulates the production of gelatinase B by T cellsand may thus impair lymphocyte infiltration into MSplaques.(11,12) However, activation of T cells by antigen-pre-senting cells (APC) generally occurs within the context of ma-jor histocompatibility complex (MHC)/T cell receptor (TCR)interactions. A recent study strongly suggested that gelatinaseB is involved in the maturation and migration of Langerhanscells,(13) the cutaneous subtype of DC. Relatively little is knownabout gelatinase B expression by DC, and it is not establishedat all whether IFN-b might modulate gelatinase B productionand activity by this type of cell.

In the present study, we document that DC, generated by cul-turing adherent peripheral blood mononuclear cells (PBMC) ingranulocyte-macrophage colony-stimulating factor (GM-CSF)and interleukin-4 (IL-4), secrete bioactive gelatinase B and thatIFN-b inhibits this production.

1Department of Immunology, Hôpital Erasme, 2Centre de Recherche Inter-Universitaire en Vaccinologie (C.R.I.V.), and 3Laboratory of His-tology, Université Libre de Bruxelles, Brussels, Belgium.

4Laboratory of Molecular Immunology, Rega Institute for Medical Research, Leuven, Belgium.

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MATERIALS AND METHODS

Culture medium and reagents

All cultures were in RPMI 1640 (BioWhittaker Europe,Verviers, Belgium) supplemented with 2 mM L-glutamine(GIBCO, Paisley, Scotland), 20 mg/ml gentamicin, 50 mM 2-mercaptoethanol, 1% nonessential amino acids (GIBCO), and10% fetal bovine serum (FBS) (BioWhittaker Europe). Recom-binant IL-4 (24 3 106 IU/mg) and recombinant GM-CSF (Leu-comax, 16 3 103 IU/mg) were kindly provided by Schering-Plough (Kenilworth, NJ). Recombinant IFN-b1a was kindlyprovided by G.J. van Daal (Serono Benelux, Den Haag, TheNetherlands). For all culture reagents, the endotoxin content ofthe final dilutions used was ,10 pg/ml as determined by a limu-lus amebocyte lysate assay (QCL-1000) (BioWhittaker Europe).

Generation of DC from peripheral blood of healthy donors

PBMC from healthy volunteers were isolated by densitycentrifugation of heparinized blood on Lymphoprep (Ny-comed, Oslo, Norway). About 15 3 106 adherent PBMC werethen cultured in 75-cm2 plastic flasks in 20 ml medium. Im-mature myeloid DC were generated by adding 16,000 IU GM-CSF and 10,000 IU IL-4 every 2 days from day 0 to the flasks,as described by Romani.(14) After 6 or 7 days of culture, DCwere harvested, washed two times in medium, and used forsubsequent experiments. The resulting cell preparation con-tained ,10% CD31, CD141, CD161, CD561, or CD191

cells, as assessed by fluorescence-activated cell sorter (FACS)analysis.

DC stimulation

All cultures were performed in medium supplemented withGM-CSF (800 IU/ml) and IL-4 (500 IU/ml). Immature DC (4 3

105 per well) in 24-well culture plate were cultured in the pres-ence or absence of lipopolysaccharide (LPS) (1 mg/ml), tumornecrosis factor-a (TNF-a) (25 pg/ml) and IL-1b (10 pg/ml)and/or IFN-b (10–104 IU/ml). For determination of gelatinaseB production and net gelatinase activity, supernatants were re-covered after 3 days of culture.

Histochemical procedures

All histochemical procedures were carried out as detailedpreviously.(15) Briefly, 5-mm thick sections were taken from im-mature DC formalin-fixed, paraffin-embedded pellets. Incuba-tion with gelatinase A-specific or gelatinase B-specific mouseantibody (Oncogen Research Products, Cambridge, MA) orwith the corresponding IgG1 monoclonal isotype (R&D Sys-tems Europe, Oxon, U.K.) was carried out at 25 6 1°C for 120min at 2 mg/ml. The extent of the specifically bound antibod-ies was demonstrated with avidin-biotin-peroxydase complex(ABC) kit reagents (Vector Labs, Burlingame, CA), with di-aminobenzidine/H2O2 as the chromogenic substrates. Counter-staining with hematoxylin concluded the processing.

Analysis of gelatinase B by zymography

Gelatinase production was analyzed by substrate zymogra-phy, essentially as described by Masure et al.(16) DC super-

natants were supplemented with an equal volume of 4% SDS-containing sample buffer, by which noncovalent complexes ofgelatinases are dissociated. This method visualizes the totalgelatinase B activity because the latent (inactive) proenzymeform is activated by the SDS and becomes detectable, like theactivated enzyme present in the sample. In addition, enzymat-ically inactive complexes of gelatinase B with tissue inhibitorsof MMP, TIMP, are also dissociated,(17) and the liberated en-zyme is detected by zymography. This implies that this methodis superior to other techniques to visualize the total gelatinasecontent but also that this technique does not yield information

BARTHOLOMÉ ET AL.496

FIG. 1. Fluorescent-activated gelatin substrate conversion(FASC) assay. (A) Distribution of autofluorescence of uncoatedpolystyrene microbeads (psmb). (B) After labeling of gelatinwith fluorescein and absorption to the microbeads, the his-togram gfpsmb is generated. (C) Incubation of the beads in sam-ple buffer generates the background histogram gfpsmbi, and (D)incubation with a sample results in a decrease of fluorescenceof the beads (sample) proportional to the net gelatinolytic ac-tivity (Fig. 2). FASC units are defined as gfpsmbi minus sam-ple values.

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on the net gelatinolytic activity of the samples. In all instances,the presence of constitutively produced gelatinase A and theinclusion of molecular weight markers served as standardiza-tion in the identification of the gelatinase B zymolysis. Scan-ning densitometry was used to quantify the gelatinolytic ac-tivity.

Fluorescent-activated substrate conversion (FASC) assay

The FASC assay has been described previously in detail bySt-Pierre et al.(18) Briefly, polystyrene microspheres, with a di-ameter of 14.6 6 1.7 mm (mean 6 SD) (Polyscience, War-rington, PA), were coated with fluorescein isothiocynate(FITC)-labeled gelatin, blocked with bovine serum albumin(BSA), and washed several times with phosphate-bufferedsaline (PBS), supplemented with 0.5% BSA and 0.05% sodiumazide (PBA). Uncoated (to measure background activity of thebeads) and gelatin-coated microspheres (to measure the maxi-mum fluorescence) were analyzed before and after incubationin cell culture medium in a FACScan flow cytometer (BectonDickinson Immunocytometry Systems Europe, St.-Niklaas,Belgium) to determine autofluorescence and maximal fluores-cence of labeled microspheres. To determine net gelatinolyticactivity, 90 ml culture supernatant of DC was supplementedwith 10 ml fluorescent gelatin-coated microsphere suspensionand incubated for 16 h at 37°C. To stop the enzyme reaction,1000 ml PBA was added and the microspheres were pellettedand washed two times in PBA. Then the microspheres were re-suspended in 500 ml PBA, and the fluorescence was measuredin the flow cytometer (Fig. 1). The stock solutions of labeledgelatin and microspheres were stored and manipulated underminimal light. As previously described,(18) the range of this as-say is broad, and the interassay variation is minimal. We ex-pressed the net gelatinase activity as FASC units by subtrac-

tion of the sample value from the maximal value. This impliesthat with increasing gelatinase activity, the FASC units will in-crease accordingly. For each assay, a standard curve of elec-trophoretically pure natural gelatinase B sample from humanneutrophils (laboratory standard(19)) yielded a linear FASC unitrelation between 30 and 300 ng gelatinase B (Fig. 2).

Statistical analysis

Statistical significance was determined using the two-tailed,paired Wilcoxon test. Friedman’s test was used to test dose-re-sponse experiment statistical significance.

IFN-b INHIBITS GELATINASE B PRODUCTION BY DC 497

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FIG. 2. FASC assay standard curve. Standard dilution seriesof electrophoretically pure human gelatinase B from humanneutrophils (laboratory standard(19)) results in a linear relationbetween 30 and 300 FASC units in which 300 FASC units cor-respond to 1000 ng gelatinase B activity. Data from one repre-sentative experiment.

FIG. 3. Immature monocyte-derived DC express gelatinaseA and B. Immunohistochemical staining of 5-mm thick sectionsfrom immature DC formalin-fixed, paraffin-embedded pellets.Counterstaining with hematoxylin (3100). (C) Strongly posi-tive staining for gelatinase B was detected in more than 80%of DC. (B) Gelatinase A staining of DC was less frequent andless intense. (A) No staining was observed when sections wereincubated in the presence of a control IgG1 mAb.

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RESULTS

Monocyte-derived DC express gelatinase B

As expected from previous experiments,(14) plastic-adherentPBMC cultured for 6 days in GM-CSF and IL-4 displayed themorphology of immature DC (data not shown), still lacking thetypical dendritic aspect of mature DC. The cells expressedCD1a, CD40, CD80, human histocompatibility leukocyte anti-gen-DR (HLA-DR), but no CD83 on their surface (data notshown). In order to demonstrate the presence of gelatinase Bin the cytoplasm of DC, immunohistochemical experimentswere conducted on sections of fixed immature DC pellets. Asshown in Figure 3, strongly positive staining for gelatinase Bwas detected in more than 80% of DC generated in vitro fromthe PBMC of 4 healthy blood donors. Gelatinase A staining ofDC was less frequent and less intense. No staining was ob-served when sections were incubated in the presence of a con-trol IgG1 monoclonal antibody (mAb). Similar results havebeen obtained in situ on Langerhans cells by Uchi et al.(20)

Monocyte-derived DC secrete bioactive gelatinase B

In zymography analysis (Fig. 4), a major 92-kDa lytic bandrepresenting gelatinase B was detected in supernatants of DCcultured for 72 h in medium containing GM-CSF and IL-4. Anadditional 65-kDa lytic band, representing gelatinase A, wasdetected in 9 of the 15 experiments. When present, zymolyticgelatinase A production remained low compared with gelati-nase B production (gelatinase B/gelatinase A optic density ra-tio 6 standard error of the mean [SEM] 5 7.67 6 2.02; p ,

0.05).Production of gelatinase A or gelatinase B or both has been

demonstrated previously in various cell types, such as neu-trophils, mast cells, macrophages, Langerhans cells, T cells, andsome invasive tumor cells. In these cell types, gelatinase B se-cretion is induced by various stimuli, such as TNF-a, IL-1b, orgrowth factors.(4,16,17,20–22) The basal production of gelatinaseB by immature DC cultured in GM-CSF and IL-4 reported heremay reflect the high level of protein synthesis usually observedin these cells.(1) In addition, GM-CSF has been demonstratedto enhance MMP secretion by monocytes, whereas IL-4 has aninhibitory effect. In most studies on gelatinase production byimmune cells, the technique of gelatin zymography was used.For instance, natural MMP-9 was purified from antigen-pre-senting monocytes(17) and from granulocytes,(19) and its regu-lation by cytokines, chemokines, and other agonists was stud-ied in these cell types(4,17,23,24) and in lymphocytes(11,12) usingzymography. However, this type of assay is not informativeabout the net gelatinolytic activity, that is, the balances betweenlatent proenzymes and activated gelatinases and between en-zymes and enzyme inhibitors. Four different types of physio-logic tissue inhibitors of MMP, TIMP, have been identified.TIMP all inhibit the active forms of MMP, but only two mem-bers of the family, TIMP-1 and TIMP-2, are secreted by mono-cytes/macrophages.(17,22,25) In these experiments, TIMP-1 pro-duction by human monocytes was five times higher thangelatinase B production.(17,22) Because the same stimuli mayhave parallel or opposite effects on the different MMP andTIMP production,(17,22,25) a novel sensitive FASC assay wasdeveloped(18) and used in the present study to complement the

zymography data from crude cell culture supernatants and todecipher whether DC produce net enzymatic activity, bioactivegelatinase. All the 15 cultures of unstimulated DC cultured 72h in GM-CSF and IL-4 demonstrated net gelatinolytic activityresulting in a 27.25% (95% confidence interval [CI]: 19.48% 235.02%; p , 0.05) decrease in fluorescence of the FITC-labeled

BARTHOLOMÉ ET AL.498

FIG. 4. IFN-b antagonizes gelatinase B production by DC. DCwere stimulated for 72 h with TNF-a/IL-1b, LPS, or IFN-b atdifferent doses or combinations, and the supernatants were ana-lyzed by gelatin zymography. Gelatinase A and B are indicatedin relation to the molecular weight marker. Gelatinase A is con-stitutively present in all the samples at the top and is visible asa lysis zone located between the molecular markers of 97 kDaand 46 kDa. Gelatinase A is not visible at the bottom becauseless sample was loaded to show the inducibility and effect ofIFN-b on gelatinase B production. Gelatinase B migrates just be-low the 97-kDa marker. IFN-b inhibited spontaneous and TNF-aIL-1b-induced DC gelatinase B production (Bottom) as wellas LPS-induced gelatinase B production (Top).

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gelatin-coated microspheres as compared with undigested mi-crospheres (Fig. 1). It is concluded that DC produce net bioac-tive gelatinase.

IFN-b inhibits DC gelatinase B production

To evaluate factors able to modify gelatinase B production,DC were cultured for 72 h in the presence of LPS, TNF-a/IL-1b, with or without IFN-b. Supernatants were tested in zy-mography analysis and FASC assay.

TNF-a/IL-1b stimulation caused a significant (p , 0.05) in-crease in gelatinase B production in zymography analysis butfailed to increase net gelatinase activity in FASC assay (Table1). LPS stimulation did not induce significant reproduciblechange in gelatinase B production or net gelatinase activity.These results contrast with the observation that TNF-a, IL-1b,

and LPS stimulate gelatinase B secretion by monocytes. Thesedata may be the result of maximal activation of gelatinase Bproduction in immature DC or the concomitant induction ofTIMP secretion, as has been shown on monocytes.(17,22)

Interestingly, we were able to show that IFN-b diminishesnot only the total zymolytic production (p , 0.05) but also thenet bioactive gelatinase produced (p , 0.05) by DC (Table 1).As demonstrated in Figure 5, the inhibitory effect of IFN-b ongelatinase B production and net gelatinase activity was dose de-pendent, maximal for 1000 IU/ml and already present at 10IU/ml. IFN-b was also able to inhibit TNF-a/IL-1b or LPS in-duction of DC gelatinase B production when present (Fig. 2).

A similar inhibitory effect of IFN-b on gelatinase B pro-duction and activity has been demonstrated on T cells and someinvasive tumour cells(12,26,27) but has not yet been studied onAPC.

IFN-b INHIBITS GELATINASE B PRODUCTION BY DC 499

TABLE 1. EFFECTS OF IFN-b AND MATURATION STIMULI ON GELATINASE B PRODUCTION BY DC

Maturation stimulia

LPS TNF-a/IL-1b IFN-b

Gelatinase B productionb 1.18 (0.77–1.59) 1.50 (1.25–1.75)c 0.78 (0.60–0.96)c

Net gelatinase activityd 0.79 (0.39–1.19) 1.20 (0.30–2.10)c 0.52 (0.22–0.82)c

aDC (4 3 105) were cultured 72 h in medium alone or in the presence of 1 mg/ml LPS, 25 pg/ml TNF-a, and 10 pg/ml IL-1b or 103 IU/ml IFN-b. Data are expressed as ratios of values obtained with treatedDC vs. DC cultured in medium alone. 95% confidence interval of at least six independent experiments isshown in parentheses.

bGelatinase B production in the supernatant fluids as measured by zymography analysis.cDouble-sided, paired, Wilcoxon exact p value , 0.05.dNet gelatinase activity in the supernatant fluids as measured by FASC analysis.

FIG. 5. Inhibition of gelatinase B expression and total gelatinase activity is dose dependent. DC generated in the presence ofGM-CSF and IL-4 were cultured for 72 h in the presence of 0–104 IU/ml IFN-b. Supernatant fluids were tested for gelatinase Bproduction by zymography analysis (A) and for net gelatinase activity by FASC assay (B). Data are given as mean ratios of theresults obtained with treated DC vs. DC cultured in medium alone (6 SEM) in four independent experiments. For both experi-ments, the exact p value, as determined with the Friedman’s test, is indicated.

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DISCUSSION

Our data indicate that IFN-b inhibits gelatinase B produc-tion by DC, a major component of the antigen-specific immunesystem. In addition to its similar effect on T cells, this inhibitoryeffect on DC gelatinase B production may lead to inhibition ofeffector cell access to the inflammatory focus. Indeed, anothertype I IFN, IFN-a, has been shown to inhibit antigen-inducedeosinophil and CD41 T cell recruitment into tissue in a murinemodel of airway late-phase reaction.(28) Similarly, inhibition ofgelatinase B has been shown recently to result in inhibition ofmigration and maturation of Langerhans cells, a particular sub-class of DC present in the epidermis, bronchi, and mucosae.(13)

The inhibition of bioactive gelatinase B production by IFN-b is of particular interest in MS, a disease in which T cellsreactive against myelin self-antigens are thought to be themain effectors of the central nervous system lesions (reviewedin ref. 29). In addition to its potential effect on cell migration,the inhibition of gelatinase B by IFN-b may have other ben-eficial effects in inflammatory diseases, such as inhibition oftissue damage, inhibition of MMP-mediated cellular releaseof TNF-a, and inhibition of extracellular antigen process-ing.(30) Some time ago, we proposed the remnant epitopes gen-erate autoimmunity concept, or REGA model, to explain thepathophysiology of MS and other autoimmune diseases in mo-lecular terms, that is, cytokines, chemokines, and the balancebetween proteases and their inhibitors.(30) In this model, ex-tracellular proteolysis, in particular by leukocyte-derivedgelatinase B, was central and suggested to anticipate T cellactivation. Cleavage of proteins, such as myelin basic protein(MBP), into immunodominant peptides by extracellular pro-teolysis and presentation of these peptides to autoreactive Tcells were suggested to trigger autoimmune responses. In ad-dition, it was suggested that disease-promoting cytokines andchemokines (IL-1b, TNF-a, IFN-g, monocyte chemotacticproteins, granulocyte chemotactic proteins) and disease-lim-iting cytokines (IL-6, transforming growth factor-b, IFN-b)act in these diseases via regulation of the protease balance.Particularly relevant, as predicted in the context of the REGAmodel and by the present study on net gelatinolytic activityin DC, is the recent finding that DC that carry HLA-DM canextracellularly load antigenic peptides.(31) In MS, such acti-vated DC or other APC may thus produce net gelatinolytic ac-tivity, which efficiently cleaves MBP into the immunodomi-nant (and encephalitogenic) peptides,(32) load these onHLA-DM, and eventually activate MBP-responsive T cells.Our study further shows that IFN-b therapy of MS may bebeneficial(9,10) by lowering the net gelatinolytic activity of DCand by decreasing the titers of the presented remnant epitopesgenerating autoimmunity. By these results, the notion that spe-cific gelatinase B inhibitors may complement the current ther-apies (IFN-b and glatiramer acetate) of MS is reinforced.

ACKNOWLEDGMENTS

This work was supported by a grant from the Charcot Foun-dation, Brussels, Belgium. E.J.B. is a recipient of a Smith KlineBeecham fellowship from the National Fund for Scientific Re-search (FNRS), Belgium. The Centre de Recherche Inter-Uni-

versitaire en Vaccinologie is sponsored by SmithKlineBeecham Biologicals and the Région Wallonne. G.O. is sup-ported by Fortis Insurances AB, Belgium, and the FNRS.

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32. PROOST, P., VAN DAMME, J., and OPDENAKER, G. (1993).Leukocyte gelatinase B cleavage releases encephalitogens from hu-man myelin basic protein. Biochem. Biophys. Res. Commun. 192,1175–1181.

Address reprint request to:Dr. E. J. Bartholomé

Department of Immunology, Hôpital ErasmeRoute de Lennik, 808

B-1070 BrusselsBelgium

Tel: 132 3 555 38 62Fax: 132 2 555 44 99

E-mail: ebarthol@ulb.ac.be

Received 2 February 2001/Accepted 20 March 2001

IFN-b INHIBITS GELATINASE B PRODUCTION BY DC 501

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