The Journal of Immunology

Human Mast Cell-Derived B ( -9) Is Regulated by Inflammatory : Role in Cell Migration1

Nick Di Girolamo,* Ikuko Indoh,* Nicole Jackson,* Denis Wakefield,* H. Patrick McNeil,* Weixing Yan,* Carolyn Geczy,* Jonathan P. Arm,† and Nicodemus Tedla2*

Mast cells are key effectors in the pathogenesis of inflammatory and tissue destructive diseases such as rheumatoid (RA). These cells contain specialized secretory granules loaded with bioactive molecules including cytokines, growth factors, and pro- teases that are released upon activation. This study investigated the regulation of MMP-9 () in human mast cells by cytokines that are known to be involved in the pathogenesis of RA. Immunohistochemical staining of synovial tissue showed abundant expression of MMP-9 by synovial tissue mast cells in patients with RA but not in normal controls. The expression, activity, and production of MMP-9 in mast cells was confirmed by RT-PCR, zymography, and Western blotting using cord blood-derived human mast cells (CB-HMC). Treatment of CB-HMC with TNF-␣ significantly increased the expression of MMP-9 mRNA and up-regulated the activity of MMP-9 in a time- and dose-dependent manner. By contrast, IFN-␥ inhibited MMP-9 mRNA and expression. The -mediated regulation of MMP-9 was also apparent in the human mast cell line (HMC-1) and in mouse marrow-derived mast cells. Furthermore, TNF-␣ significantly increased the invasiveness of CB-HMC across Matrigel-coated membranes while the addition of IFN-␥, rTIMP-1, or pharmacological MMP inhibitors signif- icantly reduced this process. These observations suggest that MMP-9 is not a stored product in mast cells but these cells are capable of producing this under inflammatory conditions that may facilitate the migration of mast cell progenitors to sites of inflammation and may also contribute to local tissue damage. The Journal of Immunology, 2006, 177: 2638–2650.

ast cells are derived from hemopoietic progenitor cells synovial tissue derived from patients with RA (8, 9). Despite the that home to tissue as committed progenitors (1–3). extensive knowledge regarding the mediators that regulate mast Maturation and differentiation of mast cells occurs in cell phenotype and number (10), factors that regulate the homing M 3 tissue in response to local production of stem cell factor (SCF) of mast cell progenitors from the bone marrow to the tissue of (4). Mast cells can also undergo significant change in number or in residence are not well-defined. phenotype during allergic or nonallergic inflammation (1, 5–9). Matrix (MMPs) are a family of neutral pro- These cells play a crucial role in IgE-dependent immune responses teolytic active against all major components of the ex- that mediate immediate hypersensitivity reactions associated with tracellular matrix. To date, 24 members have been cloned and allergic phenomena and host resistance to parasites (5, 10). They characterized in humans and are divided into four groups depend- also participate in innate immunity to bacterial infection (11) and ing on structure and preference (17). Broadly, they in- play a direct role in the pathogenesis of a mouse model of inflam- clude the , , stromelysins, and the mem- matory arthritis (12). There is considerable circumstantial evidence brane-type MMPs. Gelatinase B (92-kDa, MMP-9) and gelatinase implicating mast cells in the pathogenesis of human rheumatoid A (72-kDa, MMP-2) are the largest members of the MMP family arthritis (RA). These include the production of inflammatory (13, and are to date the only gelatinases identified. MMP docking mol- 14) and tissue destructive (15, 16) mediators. Moreover, a signif- ecules such as CD44 for MMP-9 (18) and membrane-type MMPs icant increase in mast cell numbers has been documented in human for MMP-2 (17) have been identified on the surface of various cells. Cells use these proteinases to promote local and *Inflammatory Diseases Research Unit, School of Medical Sciences, University of to enhance their invasive potential (18, 19). Proteolytically active New South Wales, Sydney, New South Wales, Australia; and †Division of Rheuma- and inducible levels of MMPs have been observed in synovial cells tology, Immunology and Allergy, Brigham and Women’s Hospital and Department of Medicine, Harvard Medical School, Boston, MA 02115 from patients with RA (20). MMP-9 is regulated posttranscriptionally at multiple levels. For Received for publication January 28, 2005. Accepted for publication May 23, 2006. example, its activity is neutralized by binding to naturally occur- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance ring tissue inhibitors of metalloproteinase (TIMP-1 to TIMP-4) with 18 U.S.C. Section 1734 solely to indicate this fact. with preferential binding observed between MMP-9 and TIMP-1 1 This work was supported by the National Health and Medical Research Council of (21). MMPs (including MMP-9) are generally synthesized and se- Australia (project grant), the Arthritis Foundation of Australia, and a Postdoctoral Fellowship from the American Arthritis Foundation. creted as latent soluble enzymes that require activation in the ex- tracellular space. Several studies have identified mast cell-derived 2 Address correspondence and reprint requests to Dr. Nicodemus Tedla, Inflammatory Diseases Research Unit, School of Medical Sciences, University of New South Wales, chymase and tryptase as potent activators of MMPs (22, 23). Sydney, New South Wales, 2052 Australia. E-mail address: [email protected] MMPs are also regulated at the level of transcription and their 3 Abbreviations used in this paper: SCF, stem cell factor; RA, rheumatoid arthritis; expression is modulated by a variety of stimuli including cytokines MMP, matrix metalloproteinase; rh, recombinant human; TIMP, tissue inhibitor of metalloproteinase; CB-HMC, cord blood derived-human mast cell; mBMMC, mouse (20, 24, 25), growth factors (26), and cell-to-cell and cell-to-matrix bone marrow-derived mast cell. interactions (27–29). Interestingly, the expression of MMP-2

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 2639 rarely varies, and it is regarded as a housekeeping-like gene in established institutional guidelines and ethics committee approval. CB- MMP biology. HMCs were established from the mononuclear cell fraction of the cord MMP-9 was first identified in (30) but these gran- blood as described (39, 40). In brief, heparin-treated cord blood was sedi- mented with 4.5% dextran solution to remove erythrocytes. Buffy coats ulocytes lack TIMP-1 (31). MMP-9 is also a product of other in- were layered onto Ficoll-Hypaque (Pharmacia) and the mononuclear cell flammatory leukocytes including T cells, macrophages, and eosin- interface was obtained after centrifugation. After repeated washes with ophils (32). Relevant to acute and chronic inflammation, MMP-9 PBS containing 5 mM EDTA, mononuclear cells were suspended at 2 ϫ 6 can cleave IL-8 and potentiate its activity as a chemoat- 10 /ml in high glucose RPMI 1640 (Invitrogen Life Technologies) con- taining 10% FBS, 2 mM L-glutamine, 0.1 mM nonessential amino acids, tractant by at least 10-fold (18). There is now compelling evidence 100 U/ml penicillin, 100 mg/ml streptomycin, 2 ␮g/ml gentamicin (all that along with the arsenal of stored serine , mast cells are from Sigma-Aldrich), and 0.2 ␮M 2-ME ((Invitrogen Life Technologies). also a major source of MMPs such as MMP-1 (33), MMP-3 (34), Cells were cultured in the presence of 100 ng/ml SCF (gift from Amgen), and MMP-9 (23, 35–37). Although there is limited evidence for 50 ng/ml IL-6 (R&D Systems), and 10 ng/ml IL-10 (R&D Systems). The the expression of MMP-9 in mast cells in rheumatoid synovium nonadherent cells were transferred every week for up to 10 wk into culture medium containing fresh cytokines. The purity and maturity of cells was (35), its regulation in RA is poorly understood. In the current assessed weekly by flow cytometry using a fluorochrome-conjugated study, we confirm MMP-9 expression in rheumatoid synovial mast mouse Ab directed against human c-kit (CD117; BD Pharmingen), by cells and for the first time, demonstrate its regulation by TNF-␣ metachromatic staining of cytospin preparations using toluidine blue, and and IFN-␥ in cord blood-derived human mast cells (CB-HMC), by immunostaining for both tryptase (DakoCytomation) and chymase (Chemicon International) using acetone-fixed cytospin preparations. Ma- bone marrow-derived mouse mast cells (mBMMC), and the human turity was defined by Ͼ95% CD117high cells, Ͼ95% toluidine blue posi- mast cell line-1 (HMC-1). Furthermore, we provide evidence im- tivity, and positive immunostaining for tryptase and chymase. Once cells plicating TNF-inducible MMP-9 in the migration of mast cells reached maturity, no other immunocytochemical or functional differences through matrix . were noted between 7- and 12-wk-old cells. Therefore, cells were used for this study when they reached Ͼ95% toluidine blue positivity rather than a specific number of weeks in culture. Materials and Methods The human mast cell leukemia cell line (HMC-1) was provided Dr. J. Patients Butterfield (Division of Allergic Diseases and Internal Medicine, Mayo Clinic, Rochester, MN) and cultured in the same medium as the CB-HMC Formalin-fixed paraffin-embedded archival synovial tissue from patients ϭ ϭ (RPMI 1640 complete medium without growth factors). Because of the with RA (n 3) and normal controls (n 3) was obtained from a tissue functional heterogeneity of the HMC-1 bulk culture, a limiting dilution collection held at the Department of Pathology, University of New South assay was performed to isolate individual HMC-1. Cells from passages 5 Wales (Sydney, Australia). Institutional human ethics committee approval to 8 of the newly propagated clones were used. was obtained for this study. Immunohistochemical studies Mouse mast cell cultures Serial sections (2–4 ␮m) of formalin-fixed paraffin-embedded synovial mBMMC were obtained by culturing primary bone marrow cells from tissue were used for immunohistochemical studies. Specific mouse IgG1 female 6- to 8-wk-old BALB/c mice (BRC) in 50% RPMI 1640 complete mAbs against MMP-9 (Ab-8) and mast cell tryptase (AA1) were purchased medium, 30% WEHI-3B supernatant (as a source of murine IL-3) and 20% from Oncogene Research Products and DakoCytomation, respectively. An 3T3 supernatant (as a source of murine SCF) (41). The nonadherent cells irrelevant mouse IgG1 negative control Ab was purchased from DakoCy- were transferred every week for up to 4 wk into fresh culture medium. The tomation. These Abs were used in a modified three-step alkaline phospha- purity and maturity of cells was assessed weekly by flow cytometry using tase staining procedure (38). In brief, paraffin-embedded adjacent sections fluorochrome-conjugated rat Ab against mouse c-kit (CD117; BD Pharm- were dewaxed and equilibrated with TBS (pH 8.0) followed by a 5-min Ag ingen) and by metachromatic staining of cytospin preparations using tolu- retrieval by microwave in 0.01 M citrate buffer (pH 6.2). The citrate buffer idine blue. Maturity was defined by Ͼ95% CD117high cells and Ͼ95% was allowed to cool, sections were re-equilibrated in TBS, then blocked toluidine blue positivity. Cells reached Ͼ95% maturity within 3–5 wk and with 20% goat serum for 20 min at room temperature. Sections were in- no differences in morphology and function were noted between 3- and cubated with anti-MMP-9 (4 ␮g/ml), anti-tryptase (5 ␮g/ml), or an isotype 5-wk cells. Cells were used when they reached Ͼ95% toluidine blue pos- control (5 ␮g/ml) Ab in 2% BSA/TBS overnight at 4°C. Sections were itivity rather than a specific number of weeks in culture. washed extensively with TBS, and then incubated with a biotinylated goat anti-mouse IgG (Vector Laboratories) for1hatroom temperature. Sec- MMP-9 production by human and mouse mast cells after tions were rinsed in TBS, and incubated with streptavidin-alkaline phos- cytokine stimulation phatase conjugate (Vector Laboratories) for 45 min at room temperature. Immunoreactivity was detected using an alkaline phosphatase substrate CB-HMC were washed twice with medium alone and resuspended at 2 ϫ (Vector Red; Vector Laboratories) and the sections were briefly counter- 106 cells/ml in assay medium (2.5% FBS/RPMI 1640 and SCF (100 ng/ stained with hematoxylin. ml)). HMC-1 and mBMMC were resuspended in the same medium and at the same density, but without SCF. Human mast cells were incubated with Morphometry 25 ng/ml TNF-␣ (R&D Systems) or 25 ng/ml IFN-␥ (Endogen) in 24-well flat-bottom Falcon plates at 37°C and 5% CO . mBMMC were incubated Overlapping images (1345 ϫ 1033 pixels), each spanning an area of ϳ1.5 2 with 25 ng/ml recombinant mouse TNF-␣ (R&D Systems), 12.5 ng/ml mm2, were taken from sections using an Olympus BX60 microscope with recombinant mouse IFN-␥ (R&D Systems), or the combination of both for a ϫ10 objective linked to Spot Advanced software version 3.5.6 for Win- 24–36 h. For some experiments, RNA was extracted and supernatants har- dows (Diagnostic Instruments). Multiple images taken from each section vested for -substrate zymography and Western blotting, and each were stitched into a single continuous image using Photoshop version 8 preparation was stored at Ϫ70°C. (Adobe Systems) software and the total surface area was measured. Adja- To assess the effects of IFN-␥ on TNF-␣-mediated induction of MMP-9, cent sections that were stained for tryptase and MMP-9 were overlapped, CB-HMC were incubated with the optimal concentration of TNF-␣ (25 rotated until they were aligned, and tryptase-positive and MMP-9/tryptase ng/ml) and increasing doses of IFN-␥ (0.005–50 ng/ml). Total RNA was double-positive cells were counted. Finally, the proportion of MMP-9-pos- extracted after 4 h for RT-PCR analysis and supernatants were collected itive mast cells from each tissue specimen was calculated. An area of 24 h after stimulation for zymography. To determine whether production of 1.56–2.43 mm2 from two to three images was sufficient to count over 100 MMP-9 from CB-HMC was due to de novo protein synthesis, cells were mast cells in RA synovium. However, it was necessary to count the entire stimulated with 25 ng/ml TNF-␣ and coincubated with varying concentra- normal synovium tissue section (5.84–7.35 mm2) to acquire ϳ100 mast tions of cycloheximide (0–0.5 ␮g/ml; Sigma-Aldrich) for 24 h. cells. Human mast cell cultures Quantitation of histamine released by CB-HMC Cord blood from human was obtained after routine Caesarian Degranulation of CB-HMC was induced after passive IgE sensitization and section from the Australian Cord Blood Bank at the Royal Women’s Hos- anti-IgE activation as described elsewhere (40). In brief, CB-HMC were pital (Sydney, Australia). Cord blood was collected in accordance with washed twice with RPMI 1640 and resuspended at 2 ϫ 106 cells/ml in 2640 INDUCTION OF MMP-9 BY TNF-␣ IN HUMAN MAST CELLS

␥ culture medium containing 100 ng/ml SCF. Cells were primed with semi- IFN- in 15-ml round-bottom Falcon tubes at 37°C and 5% CO2. Cells purified human myeloma IgE (10 ␮g/ml; Chemicon International) and IL-4 were washed once with 10 ml of binding buffer (1% BSA, 25 mM HEPES (10 ng/ml; Endogen) for 5 days. After washing with PBS containing 0.1% in RPMI 1640) then adjusted to 1.5 ϫ 106 cells/ml. Polyvinylpyrrolidone- BSA (ICN Biomedicals), cells were activated with 1 ␮g/ml rabbit anti- free membranes (10 ␮m pore, 25 ϫ 80 mm; Osmonics) were coated with human IgE Ab in 0.1% BSA/PBS for 30 min at 37°C in 5% CO2. For some a 1/6 dilution of Matrigel (Sigma-Aldrich) in 75 mM HEPES/RPMI 1640 experiments, cells were treated for 4–8 h with varying concentrations of for2hatroom temperature then 10 ␮g/ml human fibronectin (Invitrogen A23187 (Sigma-Aldrich) or 50 ng/ml phorbol ester (PMA; Sigma- Life Technologies) was applied for1hatroom temperature and allowed to Aldrich). Supernatants were harvested and stored at Ϫ70°C before hista- air dry. mine determination, zymography, and Western blot analysis (see below). Varying concentrations (10Ϫ10 to 10Ϫ8 M) of the mast cell chemoat- Some cells were collected by centrifugation, resuspended in RPMI 1640, tractant C5a (R&D Systems) or binding buffer alone in a final volume of and lysed by rapid freeze/thawing (three cycles). Histamine in the super- 30 ␮l were dispensed in triplicate into the lower well of a 48-well chemo- natant and cell pellet fractions was measured by ELISA (ICN Biomedi- taxis chamber (Neuro Probe). A suspension of cytokine-stimulated CB- cals). Percentage of histamine release was quantitated by the equation: HMC (50 ␮l) was dispensed into the upper wells of the chemotaxis cham- histamine in supernatant/(histamine in supernatant ϩ histamine in ber that was separated by a coated membrane. Chambers were ϫ pellet) 100. subsequently incubated at 37°C in a humid 5% CO2 incubator for 14 h, the membranes were removed, rinsed in PBS, and the upper surface gently Gelatin-substrate zymography scraped over with a wiper blade to remove noninvading cells. Membranes were stained with Diff Quick according the manufacturer’s instruction (Lab Zymography was performed as previously described (25, 33, 42, 43). Su- Aids), mounted on glass slides and coverslipped. Cells that had invaded to pernatants standardized for cell numbers were diluted 1/4 using serum-free the lower surface were counted under high power (ϫ400) using a light culture medium and 8 ␮l of nonreducing sample buffer (0.25M Tris-HCl microscope. Three randomly selected fields were counted from each well (pH 6.8), 10% SDS, 4% sucrose, 0.1% bromphenol blue) was added to 25 and each treatment consisted of triplicate wells giving nine counts per ␮l of diluted culture supernatant and loaded without boiling onto 10% treatment. The migration index from three independent experiments was SDS-PAGE gels containing 1 mg/ml gelatin (Sigma-Aldrich). After elec- then calculated by dividing the mean cell counts for each treatment divided trophoresis, gels were rinsed twice for 45 min in 2.5% Triton X-100 by counts from corresponding medium only control. (Sigma-Aldrich), incubated overnight at 37°C in substrate buffer (50 mM To determine whether the migration of CB-HMC through Matrigel was Tris-HCl (pH 7.4), 10 mM CaCl , and 0.02% NaN ), stained with Coo- 2 3 indeed MMP dependent, migration assays were performed in the presence massie Blue R-250 (Bio-Rad) for 1.5 h, then destained (3 ϫ 50 min) to of several MMP inhibitors. A broad-spectrum synthetic MMP inhibitor expose gelatinolytic bands. A low range molecular mass protein ladder (BB3103) was provided by Dr. A. J. Gearing (British Biotech, Oxford, (Bio-Rad) was run in adjacent lanes. MMP identity was verified by adding U.K.). Recombinant human (rh) TIMP-1 and TIMP-2 were purchased from proteinase inhibitors such as 10 mM EDTA, 1 mM 10-phenanthroline Calbiochem-Novabiochem and dexamethasone was purchased from (Sigma-Aldrich), and 2 mM 4(Ϫ2-aminoethyl) benzene-sulfonyl fluoride Sigma-Aldrich. Briefly, CB-HMC were preincubated in RPMI 1640 com- (ABSF; Sigma-Aldrich) to the substrate buffer. Gelatinolytic bands were plete medium that contained 100 ng/ml SCF and TNF-␣ (25 ng/ml) for 6 h semiquantified with the Gel Doc 2000 and the Quantity One program at 37°C in 5% CO . Cells were then washed once with 10 ml of binding (Bio-Rad). 2 buffer and adjusted to 1.5 ϫ 106 cells/ml in binding buffer containing Western blotting BB3103 (0–10␮⌴), rhTIMP-1/-2 (0–5 ␮g/ml), or dexamethasone (0–5 ␮⌴). Cells in binding medium alone were used as positive control. To Western blotting was performed as previously described (25, 33, 43). Cul- determine toxicity, a 100-␮l cell suspension containing each inhibitor at the ture supernatant from control or cytokine-stimulated CB-HMC (25 ␮l) was respective concentration was dispensed into the wells of a 96-well flat- electrophoretically separated on SDS-PAGE using 4% stacking and 10% bottom plate. Chemotaxis chambers and 96-well plates were incubated for resolving gels under nonreducing conditions. Proteins were transferred to 14 h at 37°C in 5% CO2. Membranes were rinsed, stained, and migrated cells PolyScreen polyvinylidene difluoride transfer membranes (PerkinElmer counted as described above. Supernatants from the 96-well plates were har- Life Sciences), blocked in 5% skim milk powder in TBST for 1 h, washed vested for zymographic analysis and cells were used for viability assays using briefly in TBST, then incubated with 5 ␮g/ml primary mAb directed a fluorometric, 96-well plate LIVE/DEAD Viability assay kit (Molecular against human MMP-9 (Ab-8; Calbiochem-Novabiochem) diluted in 5% Probes). BSA/TBST for1hatroom temperature. Membranes were then extensively washed in TBST (3 ϫ 5 min) and incubated with a 1/2000 dilution of Cell surface expression of C5a and c-kit on CB-HMC HRP-conjugated rabbit anti-mouse Ab (DakoCytomation) for1hatroom by flow cytometry temperature. Membranes were again washed (3 ϫ 5 min) in TBST, then ϫ 6 placed in chemiluminescent reagent for 1 min (Western Lightning; CB-HMC (2 10 /ml) incubated in RPMI 1640 complete medium con- taining 100 ng/ml SCF with or without TNF-␣ (25 ng/ml) for6hat37°C PerkinElmer Life Science) and exposed to Hyperfilm MP (Amersham Bio- ϫ sciences). A prestained low molecular mass protein ladder (Bio-Rad) was in 5% CO2 were washed with 10 ml of binding buffer and an aliquot (1 6 ϫ 6 run in adjacent lanes. 10 cells) was sampled (6-h time point). The remaining cells (1 10 cells/ml) were transferred to a 96-well flat-bottom plate in binding buffer RNA extraction and RT-PCR containing 5% FBS and incubated at 37°C in 5% CO2 for 14 h. Before staining, cells were washed with cold PBS containing 0.05% NaN3 and 1% Total RNA was extracted (RNAgents Total RNA Extraction kit; Promega) BSA (PAB buffer) and resuspended in the same buffer at 2 ϫ 106/ml. from control and cytokine-stimulated CB-HMC after 4 h. Reverse tran- Human serum (10% final) was added and cells (50 ␮l) were incubated for scription was performed using the Preamplification System for First Strand 30 min at room temperature with 5 ␮g/ml unconjugated mouse IgG2a mAb cDNA Synthesis kit (Invitrogen Life Technologies) and as previously de- to C5a R (CD88) (clone 7F3; a gift from Prof. C. Mackay, Garvan Institute scribed (25, 33, 43) using Superscript III. Aliquots (4 ␮l) of cDNA were for Medical Research, Sydney, Australia), control mouse IgG2a (5 ␮g/ml; amplified by PCR using 100 nM each of the forward 5Ј-TTC AAG GCT Serotec), saturating amounts of PE-conjugated mouse IgG1 mAb to c-kit GGG AAG TAC TG-3Ј and reverse 5Ј-TCC GGA GGG CCC CGG TCA (CD117; BD Pharmingen), or with PE-conjugated control mouse IgG1 (BD CCT-3Ј primers for MMP-9, TIMP-1, and GAPDH (43). A 2-min hot start Pharmingen). After washing in cold PAB buffer, 500 ␮l of 1% parafor- at 95oC was performed to denature the double stranded cDNA, followed by maldehyde in PBS was added to the cell suspensions and stained with 37 cycles of PCR (each cycle: 95°C, 30 s; 55°C, 30 s; 72°C, 30 s) and the conjugated Abs before storage at 4°C in the dark. Cells stained with uncon- reactions terminated with a 2-min extension at 72°C. Cycle number was jugated Abs were incubated on ice for 45 min with 10 ␮l (10 ␮g/ml) of Ј Ј predetermined so that the products generated were within the linear portion FITC-conjugated F(ab )2 goat anti-mouse IgG (F(ab )2-specific), secondary of the amplification curve. PCR products were visualized on 2% agarose Ab (Jackson ImmunoResearch Laboratories). Cells were washed twice with gels, precast with ethidium bromide and semiquantified with the Gel Doc PAB buffer, fixed with 1% paraformaldehyde in PBS, and analyzed by using 2000 and the Quantity One program (Bio-Rad). A 100-bp ladder (Invitro- a FACScan flow cytometer (BD Biosciences). A unimodal shift in fluores- gen Life Technologies) was run in adjacent lanes. cence intensity of cells stained with specific Abs compared with cells stained with the isotype-matched negative control Abs was considered positive. Migration of CB-HMC through Matrigel Statistical analysis CB-HMC were washed twice in RPMI 1640 and resuspended at 2 ϫ 106cells/ml in RPMI 1640 complete medium containing 100 ng/ml SCF. Significance of mast cell degranulation in response to A23187, PMA, and Cells were preincubated for 6 h in the same medium with or without IgE cross-linking was determined using two-tailed t tests. Statistical sig- TNF-␣ (25 ng/ml), IFN-␥ (25 ng/ml), or a combination of TNF-␣ and nificance in the activity of MMP-9 in mast cells treated with various doses The Journal of Immunology 2641

FIGURE 1. Immunohistochemical staining for MMP-9 in rheumatoid synovium. Serial sections of synovial tissue from three patients with RA (patient 1, A and B; patient 2, C and D; patient 3, E and F) were stained with Abs to mast cell tryptase (A, C, and E) and MMP-9 (B, D, and F), respectively. Cells in adjacent sections positive for MMP-9 and tryptase are identified with arrows. Serial sections of normal synovial tissue (normal 1, G and H) were stained with Abs to mast cell tryptase (G) and MMP-9 (H). Several mast cells were identified in this tissue, but most did not express MMP-9. The arrowheads in D, G, and H identify neutrophils within blood vessels that are positive for MMP-9. Insets, A neutrophil at high power (ϫ400) which is negative for mast cell tryptase (A) but positive for MMP-9 (B). 2642 INDUCTION OF MMP-9 BY TNF-␣ IN HUMAN MAST CELLS

Table I. Proportion of MMP-9-positive mast cells in synovial tissue of RA patients and control subjectsa

Tryptase-Positive % MMP-9-Positive Total Area Counted Subjects Mast Cells Mast Cells (mm2) Mast Cells/mm2

RA1 118 66.1 1.77 67 RA2 110 55.5 2.43 45 RA3 102 74.5 1.56 65 N1 91 2.2 7.35 12.4 N2 84 1.2 5.84 14.4 N3 97 0 9.01 10.8

a RA, Synovial tissue obtained from rheumatoid arthritis; N, normal synovial tissue obtained from patients undergoing postinjury knee surgery. of TNF-␣ or IFN-␥ at multiple time points was determined using the demonstrated substantially more tryptase-positive mast cells in Kruskal-Wallis test followed by Dunn’s multiple comparison posttest. RA synovium with an average of 57.3 Ϯ 12 cells/mm2 com- Changes in migration between treatment groups were analyzed using pared with 13.0 Ϯ 1.8 cells/mm2 in normal synovium (Table I). ANOVA with Tukey’s posttest. Modulation of after treat- ment with MMP-9 inhibitors was determined using a two-tailed t test. A There were also higher proportions of MMP-9-positive mast value of p Ͻ 0.05 was considered significant. cells in RA tissue (65.4 Ϯ 9.5%) compared with normal syno- vium (1.1 Ϯ 1.1%) (Table I). Results Production of MMP-9 by human mast cells in vivo Characterization of CB-HMCs Upon stimulation, mast cells release a battery of enzymes that are To confirm expression of MMP-9 by human mast cells, primary generally stored in specific granules. In addition to the well-char- mast cells were propagated from cord blood and characterized. acterized serine proteases, previous studies have identified mem- These cells displayed typical morphological, phenotypic, and bers of the MMPs in these cells (22, 33–37, 44). Initially, it was functional characteristics of mast cells including dense granular our intention to confirm these primarily in vitro observations in cytoplasm (Fig. 2A), intense metachromatic staining (Ͼ95%) vivo, using synovial tissue obtained from patients with RA by with acidic toluidine blue (Fig. 2A), and abundant expression immunohistochemistry. Staining for MMP-9 (Fig. 1, B, D, and F) (Ͼ97%) of tryptase (Fig. 2B) and 95–99% chymase positively was observed in tryptase-positive (Fig. 1A, C, and E) mast cells in (Fig. 2C). Flow cytometric analysis demonstrated high intensity the RA synovium whereas in normal human synovium, despite the cell surface expression for c-kit/CD117 on Ͼ95% of these cells presence of several tryptase-positive mast cells (Fig. 1G), there (Fig. 2D). was limited immunoreactivity for MMP-9 in these cells (Fig. 1H). MMP-9 was also identified in synovial lining cells, mac- Production of MMP-9 by CB-HMC rophages (data not shown) and intravascular neutrophils in RA CB-HMCs were used to determine whether MMP-9 is stored in tissue (Fig. 1, B, inset, D, arrowhead) and normal synovium mast cell granules or whether it is newly synthesized and se- (Fig. 1H, arrowhead). Quantitative analysis of tissue sections creted upon mast cell activation. To test the former hypothesis,

FIGURE 2. Characterization of hu- man cord blood-derived mast cells. Cy- tospins from 8-wk-old CB-HMC were stained metachromatically with tolu- idine blue (A) or histochemically with Abs to mast cell tryptase (B) and chy- mase (C). Flow cytometric analysis on cell suspension (D) was also performed to show surface c-kit (CD117) expres- sion. Results are representative of over 10 independent mast cell cultures from different donors. The Journal of Immunology 2643 degranulation of mast cells was induced by cross-linking of the supernatants obtained from the mast cells treated with PMA or high-affinity IgE receptor with anti-IgE for 30 min at 37°C. A23187, indicative of their degranulation (Fig. 3D). Hence, de- Although this treatment caused significant degranulation as granulation of mast cells in response to PMA, A23187, and measured by histamine release (Fig. 3C), no difference was IgE-cross linking in the absence of any significant increase in noted in the level of MMP-9 activity either in the supernatants MMP-9 production suggests MMP-9 is predominantly not a or lysates (Fig. 3A). To confirm this observation, experiments stored product. This is consistent with the lack of MMP-9-pos- were performed using other agents (A23187 and PMA) that are itive cells in normal synovial tissue (Fig. 1, G and H). known to cause the release of prestored mediators from mast cells. Activation of mast cells with PMA or A23187 ( Cytokine regulation of mast cell-derived MMP-9 ionophore) did not alter the relative production of MMP-9 even TNF-␣ plays a key role in the pathogenesis of RA and is cur- when cells were left in these agents for up to 8 h (Fig. 3B). rently a therapeutic target in this and other inflammatory dis- However, a significant amount of histamine was detected in the eases (45). TNF-␣ positively regulates expression of MMPs that

FIGURE 3. Gelatinolytic activity and histamine re- lease by CB-HMC. Cell supernatants and lysates were harvested after CB-HMC activation (30 min) with IgE and anti-IgE or IgE alone (control), neat supernatants, and ly- sates used for zymography (A). CB-HMC in RPMI 1640 medium containing 2.5% FBS and 100 ng/ml SCF were incubated without (control) or with 50 ng/ml PMA or A23187 (0.5 or 5 ␮M) for 4 or 8 h and neat culture su- pernatants were analyzed by zymography (B). Mast cell degranulation was measured by histamine release after ac- tivation of cells with IgE and anti-IgE for 30 min (C)or treatment of cells with PMA or A23187 (D). Results are p Ͻ ,ء .representative of three independent experiments p Ͻ 0.01 when compared with corresponding ,ءء ;0.05 controls. 2644 INDUCTION OF MMP-9 BY TNF-␣ IN HUMAN MAST CELLS are responsible for tissue destruction that characterizes RA. buffer containing EDTA (metal chelator) or 1,10-phenanthroline (an Therefore, we determined whether this cytokine induced the MMP inhibitor). Both agents abolished all activity associated with expression of MMP-9 in mast cells that are typically present in MMP-2 and -9 (data not shown, Refs. 42 and 43)), whereas the serine rheumatoid synovium and are believed to play a key role in the inhibitor ABSF had no effect on the activity of MMP-2 and pathogenesis of the disease. Treatment of CB-HMCs with -9 (data not shown). Moreover, stimulation with TNF-␣ not only in- TNF-␣ caused significant up-regulation of MMP-9 in a time- creased expression of the 92-kDa band, but also induced the appear- dependent manner (peak 18 h) (Fig. 4). By contrast, treatment ance of a minor 83-kDa component (Fig. 4A, 24- to 72-h arrows; Fig. of these cells with IFN-␥ significantly suppressed MMP-9 pro- 5A, arrow), a size typical of activated MMP-9. Western blotting con- duction (Fig. 4A) with the greatest decrease noted at the 24-h firmed the identity of the 92-kDa band as MMP-9 that was increased time point (Fig. 4B). This is consistent with previous studies after treatment with TNF-␣ (Fig. 5D). An immunoreactive band at 83 that have demonstrated the down-regulation MMP-9 by IFN-␥ kDa that corresponds to the active form of MMP-9 was observed in macrophages (46, 47). following TNF-␣ stimulation (Fig. 5D). Interestingly, TIMP-1, a nat- Following the kinetic studies, CB-HMCs were treated for ural inhibitor of MMP-9 protein, was not detected in the same super- 24 h with varying concentrations of TNF-␣ (0–50 ng/ml) and natants by Western blotting (data not shown) suggesting that activa- supernatants analyzed by zymography. MMP-9 production was tion of mast cells by TNF-␣ might be biased toward the process of significantly induced by TNF-␣ in a dose-dependent manner, increased tissue permeability and degradation. while MMP-2 remained unchanged (Fig. 5A). Maximum To determine whether MMP-9 production in CB-HMC was due MMP-9 production was observed when cells were stimulated to de novo protein synthesis, cells were stimulated with 25 ng/ml with 5–50 ng/ml TNF-␣ (Fig. 5C). Conversely, when CB- TNF-␣ and coincubated with varying concentrations of cyclohex- HMCs were treated with IFN-␥, constitutive MMP-9 levels sig- imide (0–0.5 ␮g/ml). MMP-9 was dose-dependently inhibited by nificantly decreased (Fig. 5B), while MMP-2 was not affected. cycloheximide and this was visualized by a decrease in intensity of Maximum inhibition was observed with 5–50 ng/ml IFN-␥ this proteinase (Fig. 6A). Inhibition of MMP-9 was statistically (Fig. 5C). significant with 0.125–0.5 ␮g/ml cycloheximide (Fig. 6B). To confirm the identity of the 92-kDa gelatinolytic band as a The effects of IFN-␥ on the TNF-␣-mediated induction of metalloproteinase, zymograms were incubated in substrate MMP-9 were examined by stimulating cells with 25 ng/ml TNF-␣

FIGURE 4. Time course production of MMP-9 in cytokine-stimulated CB-HMC. CB-HMC in RPMI 1640 containing 2.5% FBS and 100 ng/ml SCF were stimulated with either 25 ng/ml TNF-␣ or 25 ng/ml IFN-␥ and supernatants were collected at the indicated time points for zymographic analysis (A). Small arrows in A identify active MMP-9 (83 kDa) evident at 24–72 h after TNF-␣ stimulation. B, The intensity of the lytic bands from three independent experiments was measured and the fold change in intensity was calculated by dividing values from the different treatments to the values from corresponding untreated control. Bars represent mean and SE of three experiments and statistical analysis for each time point performed using Kruskal-Wallis test followed .p Ͻ 0.01 compared with untreated control ,ءء ;p Ͻ 0.05 ,ء .by Dunn’s posttest The Journal of Immunology 2645

FIGURE 6. Inhibition of MMP-9 by cycloheximide. CB-HMC in RPMI 1640 containing 2.5% FBS and 100 ng/ml SCF were stimulated with 25 ng/ml TNF-␣ in the presence of increasing concentrations of cyclohexi- mide. Supernatants were collected 24 h after treatment and analyzed by zymography (A). B, The intensity of the lytic bands from three independent experiments was measured and percentage of MMP-9 activity for each treatment relative to corresponding cycloheximide untreated control was calculated. Bars represent mean and SE of three experiments and statistical analysis for each dose performed using Kruskal-Wallis test followed by p Ͻ 0.01 compared with untreated ,ءء ;p Ͻ 0.05 ,ء .Dunn’s posttest control.

modest (1.7-fold) decrease in the TNF-␣-induced expression of MMP-9 (data not shown). TIMP-1 mRNA was detected in CB- HMC but levels were not affected by TNF-␣ and/or IFN-␥. Our observations in CB-HMC further corroborated HMC-1 and mBMMC. Treatment of HMC-1 or mBMMC with TNF-␣ induced the production of MMP-9 in these cells (Fig. 8) and IFN-␥ com- pletely abrogated TNF-␣-mediated up-regulation of this enzyme in both mast cell types (Fig. 8, A and B, lane 4). MMP-9 activity was not detected in supernatants derived from control or IFN-␥-treated FIGURE 5. Dose-dependent modulation of MMP-9 by cytokines. CB- HMC-1 or mBMMC, suggesting that these cells produce lower HMC in RPMI 1640 containing 2.5% FBS and 100 ng/ml SCF (control) constitutive levels of this protease compared with CB-HMC. Al- were cultured with varying concentrations of TNF-␣ or IFN-␥ and super- ternatively, cytokines such as SCF, IL-6, and/or IL-10 that were natants collected after 24 h for zymography at a dilution of 1/4 (A and B). used to maintain CB-HMC in culture may be responsible for the C, The intensity of the lytic bands from three independent experiments was higher basal expression of MMP-9 in these cells. measured and the fold change in intensity was calculated by dividing val- ues from the different treatments to the values from corresponding un- Invasion of CB-HMC through Matrigel is mediated by MMP-9 treated control. Bars represent mean and SE of three experiments and sta- and not by C5aR up-regulation tistical analysis for each dose performed using Kruskal-Wallis test -p Ͻ 0.05 compared with untreated control. Mast cells are generally found in abundance at sites of inflamma ,ء .followed by Dunn’s posttest D, Western blot analysis of culture supernatant obtained from cells treated tion. The means by which the progenitors of these cells traverse the for 24 h with TNF-␣ (25 ng/ml) or IFN-␥ (25 ng/ml). vascular endothelium and migrate through the surrounding con- nective tissue is poorly understood. However, evidence now sug- gests that MMPs may facilitate the extravasation, migration, and in the presence of varying amounts (0–50 ng/ml) of IFN-␥. Treat- recruitment of inflammatory cells from blood vessels to the sur- ment of cells with IFN-␥ caused inhibition of TNF-␣-mediated rounding connective tissue (48, 49). MMP-9 produced by activated MMP-9 production in a dose-dependent manner (Fig. 7). mast cells may play such a role. Migration of cytokine-treated The effect of TNF-␣ and IFN-␥ on the expression of MMP-9 CB-HMC through a Matrigel-coated membrane was investigated. mRNA was studied in cells treated with optimal concentrations of Dose-dependent migration of mast cells toward C5a was observed these cytokines for 4 h. RNA was analyzed by RT-PCR using irrespective of the cytokine treatment (Fig. 9A). However, the gene-specific primers for human MMP-9. Consistent with the pro- highest number of invading cells was observed when cells were tein data, treatment of cells with TNF-␣ induced MMP-9 mRNA pretreated with TNF-␣. This was statistically significant at all by 2.4-fold as compared with untreated controls (data not shown), doses of C5a ( p Ͻ 0.01) when compared with cells that were whereas coincubation of cells with TNF-␣ and IFN-␥ caused a preincubated in basal medium containing SCF alone. Numbers of 2646 INDUCTION OF MMP-9 BY TNF-␣ IN HUMAN MAST CELLS

FIGURE 8. Induction of MMP-9 by TNF-␣ in BMMC and HMC-1. mBMMC (A) and HMC-1 (B) cells were cultured under control conditions (lane 1), exposed to 25 ng/ml human or mouse TNF-␣ (lane 2), 25 ng/ml human or mouse IFN-␥ (lane 3) or a combination of TNF-␣ and IFN-␥ (lane 4). Supernatants were harvested after 36 h and assayed for MMP-9 without dilution by gelatin zymography. Treatment of the mBMMC and HMC-1 cells with TNF-␣ induced a 92-kDa lytic band corresponding to the expected size for MMP-9.

␣), a cytokine strongly linked to the pathogenesis of RA (45). The FIGURE 7. Inhibition of TNF-␣-mediated production of MMP-9 by ␥ TNF-mediated up-regulation of MMP-9 was strongly abrogated by IFN- . CB-HMC in RPMI 1640 containing 2.5% FBS and 100 ng/ml were ␥ ␣ treated with 25 ng/ml TNF-␣ in the presence of varying concentrations of IFN- . A similar pattern of regulation of MMP-9 by TNF- and ␥ IFN-␥ for 24 h and supernatants were analyzed by zymography (A). B, The IFN- has previously been reported in human macrophages (47), intensity of the lytic bands from three independent experiments was mea- however its regulation by TNF-␣ and IFN-␥ in primary human sured and percentage of MMP-9 activity for each treatment relative to mast cells (CB-HMC) is novel. Although the production of corresponding IFN-␥-untreated control was calculated. Bars represent MMP-9 by human mast cells has been reported (23, 33–37, 44), we mean and SE of three experiments and statistical analysis for each dose provide substantial evidence to demonstrate that MMP-9 is pri- p Ͻ marily not a stored product. Other investigators have speculated ,ء .performed using Kruskal-Wallis test followed by Dunn’s posttest -p Ͻ 0.01 compared with untreated control. that latent MMP-9 coexists with histamine and tryptase in spe ,ءء ;0.05 cialized secretory granules within mast cells (37). In that par- invading cells in samples preincubated with IFN-␥ were similar to ticular study, the HMC-1 cell line was used and cell-to-cell those cultured in basal medium containing SCF, indicating that contact with lymphocytes induced MMP-9. Given that MMP-9 IFN-␥ did not alter invasion of CB-HMC through matrix compo- protein was first detected at 6 h and peaked at 22 h, this sug- nents. However, the combination of IFN-␥ and TNF-␣ signifi- gested de novo synthesis and not the release of preformed pro- cantly ( p Ͻ 0.01) reduced cell invasion induced by TNF-␣ to tein as the authors of that study alluded (37). In CB-HMC, control levels (Fig. 9A). The increased migration of TNF-treated enhanced production of MMP-9 was observed at6hand cells toward C5a was not due to up-regulation of C5aR, as its reached maximum levels by 18–24 h following TNF-␣ stimu- constitutive expression was not altered by pretreatment of cells lation (Fig. 4). Moreover, no additional MMP-9 was released with TNF-␣ for6horsubsequent withdrawal of TNF-␣ for 14 h following mast cell degranulation (Fig. 3, A and B) and CB- (Fig. 9B). HMC lysates displayed low to undetectable levels of MMP-9 To confirm whether the increased invasion of mast cells through activity compared with supernatants derived from the same the reconstituted basement membrane after treatment with TNF-␣ cells (Fig. 3A). Furthermore, the TNF-␣-mediated induction of was mediated by MMPs, TNF-␣-primed CB-HMC were treated MMP-9 in CB-HMC was abrogated by cycloheximide (Fig. 6), with MMP inhibitors throughout the duration of the invasion as- strongly supporting the notion that primary human mast cells syn- say. Treatment of cells with broad spectrum MMP inhibitors, thesize and secrete MMP-9 when appropriately stimulated. Inter- BB3103 (Fig. 10A) and dexamethasone (Fig. 10B), or specific in- estingly, treatment of CB-HMC with TNF-␣ for 6 h was sufficient hibitors including rhTIMP-1 (Fig. 10E) or rhTIMP-2 (Fig. 10G) to induce MMP-9 production long (14 h) after its removal (Fig. 10, caused dose-dependent abrogation of cell invasion through the re- B, D, F, and H, lane 1). Synthesis and secretion of proteins by constituted basement membrane by up to 80% (range 20–80%). activated mast cells without the release of other preformed medi- The viability of the cells following treatments with the various ators has previously been documented (50). concentrations of inhibitors was consistently Ͼ95%, confirming The induction of MMP-9 and MMP-2 in CB-HMC and that the effect of these mediators on cell migration was not due to HMC-1 after PMA stimulation was reported (35). However, we toxicity. Zymographic analysis of supernatants obtained from the did not observe induction of MMP-9 following PMA stimula- TNF-␣-primed cells showed that BB3103 (Fig. 10B), rhTIMP-1 tion of CB-HMC (Fig. 3). These differences may be attributed (Fig. 10F), or rhTIMP-2 (Fig. 10H), despite their strong inhibition to modifications in culture conditions, level of mast cell matu- of cell invasion, did not inhibit the production of MMP-9 by these rity and/or incubation times with PMA. In a previous investi- cells. In contrast, treatment of these cells with dexamethasone gation, we observed potent induction of MMP-9 in HMC-1 and caused dose-dependent down-regulation of MMP-9 production by canine BR mastocytoma cells after a 48-h incubation with this ϳ2-fold as displayed by the decrease in the intensity of the gela- agent (33). Because TNF-␣ is one of the key cytokines in RA, tinolytic band (Fig. 10D, lane 4). synovial tissue from patients with RA was used to investigate in vivo expression of MMP-9 in mast cells. In addition to various Discussion other cell types, MMP-9 was colocalized in a large proportion In this study, we have demonstrated the induction of MMP-9 in (but not all) of tryptase-positive mast cells in diseased tissue primary human mast cells by a proinflammatory cytokine (TNF- (Fig. 1, A–F; Table I), whereas, MMP-9 reactivity was scant in The Journal of Immunology 2647

in mast cells from diseased human synovial tissue, it was also detected in mast cells found in normal human skin and (35). Yet other studies have not detected MMP-9 despite the presence of mast cells in normal skin (44). The above dispari- ties in the results regarding the expression of MMP-9 in normal tissue may be attributed to differences in Abs, immunohisto- chemical technique, processing of the tissue and/or site from which the tissue was obtained. Studies have shown that mast cell-derived MMP-9 can be down-regulated (36) or up-regulated (52) by SCF depending on the type of mast cells. Tanaka et al. (36) demonstrated down- regulation of MMP-9 in bone marrow-derived mouse mast cell progenitors, whereas, Fang et al. (52) demonstrated induction of MMP-9 in canine BR mastocytoma cells treated with SCF. In the current study, CB-HMCs were cultured in the presence of SCF and this may have accounted for the substantial amount of constitutive MMP-9 (Fig. 4). This is supported by the increase in production of MMP-9 in cells cultured over 6–72 h (Fig. 4). In contrast, no basal production of MMP-9 was evident in HMC-1 or mBMMC, neither of which requires rSCF for their maintenance in culture (Fig. 8). We also showed that IFN-␥ inhibited constitutive (Figs. 4 and 5) and TNF-␣-mediated in- duction of MMP-9 in CB-HMCs (Fig. 7). The inhibitory effects of IFN-␥ come as no surprise as IFN-␥ suppresses the TNF-␣-mediated induction MMP-9 (46, 47) and MMP-1 (53) in tumor cells, primary macrophages, and in synovial fibroblasts. Although, further work is required to determine the mechanism in CB-HMCs: this inhibitory effect may be mediated via STAT-1 or via caspase 8-independent as described in tumor cells (46) and synovial fibroblasts (54). In this study, we have shown that the significant increase in migration of CB-HMC through reconstituted basement membrane was associated with the TNF-␣-mediated induction of MMP-9 but not C5aR in these cells (Fig. 9). Furthermore, the TNF-␣-mediated increase in migration was abrogated by coincubation with IFN-␥, which also inhibited MMP-9 production in these cells. These re- sults suggest that MMP-9 might be important for the transmigra- tion of human mast cell progenitors across a basal lamina or/and connective tissue matrix. To investigate whether this was indeed an MMP-related phenomenon, a selection of MMP inhibitors was FIGURE 9. Invasion of CB-HMC through matrix-coated membranes. included in this model. We observed that the synthetic hydroxamic A, Invasion of CB-HMC through matrix protein-coated membranes was acid derivative (BB3103) and the corticosteroid dexamethasone performed on untreated cells or cells that were preincubated with TNF-␣, significantly reduced the TNF-␣-mediated increase in CB-HMC ␥ ␣ ␥ IFN- , or the combination of TNF- and IFN- . Cells in three high power invasion (Fig. 10, A and C). Similarly, the addition of specific fields were counted for each well from triplicate wells for each cytokine natural inhibitors (TIMP-1 and -2), abrogated cell invasion by treatment and each C5a concentration. Mean migration index from three independent experiments was then obtained by dividing the number of cells 80 and 60%, respectively (Fig. 10, E and G). Importantly, both that have migrated toward the different concentrations of C5a to the cells synthetic MMP inhibitors and the TIMPs have been reported to that have migrated to medium alone. A significantly higher number of display anti-inflammatory effects (55). Their mechanism of ac- cells invaded through the matrix protein after treatment with TNF-␣ com- tion seems to be at the posttranslational level as they bind either -the latent or the zymogen form of the MMP molecule. In con ,ءء ;p Ͻ 0.05 ,ء) ␥-pared with control cells or cells pretreated with IFN p Ͻ 0.01). The addition of IFN-␥ to the TNF-␣-treated cells significantly trast, dexamethasone has a direct effect on MMP-9 production ϩ Ͻ ϩϩ Ͻ inhibited TNF-induced migration of cells ( , p 0.05; , p 0.01). B, (Fig. 10D). ␣ ␣ Pretreatment of cells with TNF- for6h(a and b) or removal of TNF- In general, most MMP-producing cells also secrete naturally thereafter for14 h (c and d) did not change surface expression of C5aR or occurring counterregulatory molecules, the TIMPs. TIMPs prefer- c-kit (regular line histogram) when compared with untreated cells (bold histogram). Histograms on the left of each diagram show the corresponding entially bind to and deactivate MMPs with differing affinities. Al- isotype-matched negative controls. though we were able to demonstrate low levels of constitutive TIMP-1 gene expression (data not shown), we were unable to de- tect TIMP-1 protein by Western blotting. Furthermore, neither the normal synovium despite the presence of numerous mast cells gene nor the protein was regulated by TNF-␣ or IFN-␥. Previ- (Fig. 1, G and H; Table I). These data suggest that mast cell- ously, we (33) and others (37) observed low to undetectable levels derived MMP-9 may be induced during an inflammatory re- of TIMP-1 expression in HMC-1 cells. In other in vitro mast cell sponse. Others have shown similar in vivo results in humans models, kit ligand (SCF) induced MMP-9 but not TIMP-1 (52). (35) and mice (51). Interestingly, while MMP-9 was identified Interestingly, a smaller than expected (Ͻ14 kDa) immunoreactive 2648 INDUCTION OF MMP-9 BY TNF-␣ IN HUMAN MAST CELLS

FIGURE 10. Inhibition of invasion by TNF-␣-primed CB-HMC through matrix components with MMP inhibi- tors. Invasion of CB-HMC toward C5a (10Ϫ8 M) through Matrigel-coated membranes was performed on cells pretreated with TNF-␣ and incubated with increasing amounts of synthetic (A, BB3103), steroid (C, dexametha- sone), or natural MMP inhibitors (E, TIMP-1; G, TIMP-2). Cells in three high power fields were counted for each well and results are presented as the mean cell count Ϯ SD of triplicate wells for each treatment. The effect of each inhibitor on MMP-9 levels was evaluated by gelatin zymography (B, D, F, and H). Inhibition of migration was determined using a two-tailed t ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .test p Ͻ 0.001.

band corresponding to TIMP-1 was detected and attributed to pro- sig et al. (61) recently showed that MMP-9 induced in bone cessing and deactivation of this inhibitor by mast cell-derived chy- marrow cells caused release of SCF thereby permitting the mase (56). It is possible that TIMP-1 protein is degraded by chy- transfer of endothelial and hemopoietic stem cells from a qui- mase and tryptase that are abundant in these cells (Fig. 2, B and C). escent to a proliferative niche. This is an essential prerequisite Conversely, mast cell-derived serine proteases such as chymase for the recruitment and maturation of progenitor cells from the are known to convert the inactive pro-MMP-9 to active MMP-9 bone marrow to the tissue of residence (61). In MMP-9 knock- (56). out mice, release of SCF and motility of endothelial and hemo- Along with the many known functions of MMP-9 in wound poietic stem cells are impaired. Bone marrow ablation in these healing, connective , , and leu- animals resulted in failure of hemopoietic recovery and in- kocyte migration, several additional key functions are emerg- creased mortality (61). ing. MMP-9 can cleave the precursor form of IL-1␤ to an active Despite the extensive knowledge regarding proteins that regu- molecule and IL-8 is truncated at the N terminus by the same late mast cell phenotype and numbers in tissue, factors that regu- enzyme, causing amplification of its activity as a neutrophil late the homing of mast cell progenitors from the bone marrow to chemoattractant (57). Thus, specific inhibition of MMP-9 may the tissue of residence are not fully understood. A critical role ␣ ␤ result in anti-inflammatory effects, a proposal confirmed in for adhesion molecules such as 4 7 in directing tissue-selec- MMP-9 knockout mice in which deficiency in this enzyme pro- tive homing of mast cell progenitors to the peripheral tissue has tected mice from developing autoimmune diseases (58, 59) and been shown (62). However, the mechanisms on how mast cell prolonged delayed-type hypersensitivity responses (60). Heis- progenitors move within the usually dense connective tissue are The Journal of Immunology 2649 unclear. In this study, we have demonstrated increased migra- 18. Yu, Q., and I. Stamenkovic. 2000. Cell surface-localized matrix metalloprotein- ␤ tion of CB-HMC through reconstituted basement membrane ase-9 proteolytically activates TGF- and promotes tumor invasion and angio- genesis. Genes Dev. 14: 163–176. that was associated with increased production of MMP-9 (Figs. 19. Baumann, P., P. Zigrino, C. Mauch, D. Breitkreutz, and R. Nischt. 2000. Mem- 9A and 10). Thus, MMP-9 produced by mast cell progenitors brane-type 1 matrix metalloproteinase-mediated progelatinase A activation in non-tumorigenic and tumorigenic human . Br. J. Cancer 83: may enable these cells to migrate through connective tissue and 1387–1393. release SCF by a membrane cleavage mechanism thereby gen- 20. Mengshol, J. A., S. M. Kimberlee, and C. E. Brinckerhoff. 2002. Matrix metal- erating conditions that sustain mast cell maturation and differ- loproteinases as therapeutic targets in arthritic diseases. Arthritis Rheum. 46: 13–20. entiation. It is possible that the significant increase in mast cells 21. Brew, K., D. Dinakarpandian, and H. Nagase. 2000. Tissue inhibitors of metal- observed during allergic or nonallergic inflammation (1, 5, 6, 8, loproteinases: , structure and function. Biochim. Biophys. Acta 1477: 9) might be due to TNF-␣-induced MMP-9 production that fa- 267–283. 22. Lohi, J., I. Harvima, and J. Keski-Oja. 1992. Pericellular substrates of human cilitates migration of mast cell progenitors and causes local mast cell tryptase: 72,000 dalton gelatinase and fibronectin. J. Cell. Biochem. 50: release of SCF. This suggestion is supported by our demonstra- 337–349. tion of increased numbers of MMP-9-positive mast cells in in- 23. Fang, K. C., W. W. Raymond, S. C. Lazarus, and G. H. Caughey. 1996. Dog mastocytoma cells secrete a 92-kD gelatinase activated extracellularly by mast flamed synovial tissue. Modulation of MMP-9 production by cell chymase. J. Clin. Invest. 97: 1589–1596. mast cells might therefore be a useful therapeutic strategy to 24. Ries, C., and P. E. Petrides. 1995. Cytokine regulation of matrix metalloprotein- minimize the inflammatory reaction in RA. ase activity and its regulatory dysfunction in disease. Biol. Chem. Hoppe Seyler 376: 345–355. 25. Di Girolamo, N., P. McCluskey, A. Lloyd, M. T. Coroneo, and D. Wakefield. Acknowledgments 2000. Expression of MMPs and TIMPs in human pterygia and cultured pterygium epithelial cells. Invest. Ophthalmol. Vis. Sci. 41: 671–679. We acknowledge Dr. John Hunt (Department of Pathology, University of 26. Vaday, G. G., H. Schor, M. A. Rahat, N. Lahat, and O. Lider. 2001. Transforming New South Wales) for providing us with normal and diseased synovial growth factor-␤ suppresses tumor necrosis factor ␣-induced matrix metallopro- tissue, Dr. Taline Hampartzoumian for her technical and editorial advice, teinase-9 expression in monocytes. J. Leukocyte Biol. 69: 613–621. and Paul Halasz for his assistance in histomorphometry. 27. Lacraz, S., P. Isler, E. Vey, H. G. Welgus, and J. M. Dayer. 1994. Direct contact between T lymphocytes and monocytes is a major pathway for induction of metalloproteinase expression. J. Biol. Chem. 269: 22027–22033. Disclosures 28. Aoudjit, F., E. F. Potworowski, and Y. St-Pierre. 1998. 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