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Expression of membrane type-4 matrix metalloproteinase (metalloproteinase-17) by human eosinophils
The International Journal of Biochemistry & Cell Biology 35 (2003) 1667–1673
Expression of membrane type-4 matrix metalloproteinase (metalloproteinase-17) by human eosinophils Marie-Christine Gauthier, Christine Racine, Claudine Ferland, Nicolas Flamand, Jamila Chakir, Guy M. Tremblay, Michel Laviolette∗ Unité de Recherche en Pneumologie, Centre de Recherche de l’Hˆopital Laval, 2725, Chemin Ste-Foy, Institut Universitaire de Cardiologie et de Pneumologie de l’Université Laval, Sainte-Foy, Que., Canada G1V 4G5 Received 24 January 2003; received in revised form 20 March 2003; accepted 25 March 2003
Abstract Circulating eosinophils need proteinases to mediate a spatially limited and orientated digestion of the extracellular matrix and to migrate into tissue. Moreover, proteinases are likely involved in tissue remodeling, a crucial feature of chronic diseases including asthma. Eosinophils express matrix metalloproteinase (MMP)-9, which is increased upon stimulation with TNF-␣. Other MMPs, the membrane type (MT)-MMPs, likely play a major role in cell invasion and tissue remodeling. MT4-MMP was identified in peripheral blood leukocyte preparations, but it is not known whether eosinophils express MT4-MMP. We investigated the expression of MT4-MMP and its modulation by TNF-␣ in purified human blood eosinophils. The constitutive expression of MT4-MMP mRNA was detected by RT-PCR in unstimulated eosinophils, lymphocytes, and monocytes, but not neutrophils. Stimulation of eosinophils with TNF-␣ increased MT4-MMP mRNA expression. This effect appeared at 4 h and reached a maximum at 8 h of incubation. MT4-MMP protein was detected in freshly isolated blood eosinophils by Western blotting and immunocytochemistry. TNF-␣ increased expression of the MT4-MMP protein. MT4-MMP protein was also detected in nasal polyp eosinophils by immunohistochemistry. In conclusion, eosinophils constitutively express MT4-MMP, which is increased upon stimulation with TNF-␣. Consequently, MT4-MMP may be directly involved in the degradation of extracellular matrix components and/or modulate the activity of other proteins implicated in eosinophil migration and tissue remodeling. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Expression; Membrane type-4 matrix metalloproteinase; Human eosinophils
1. Introduction Tissue accumulation of eosinophils is a prominent feature of chronic conditions such as allergy, asthma and nasal polyposis (Jordana, Dolovich, Ohno, Abbrevitions: ECM, extracellular matrix; MBP, major basic protein; MMP, matrix metalloproteinase; MT-MMP, membrane type-matrix metalloproteinase; uPAR, urokinase plasminogen activator receptor ∗ Corresponding author. Tel.: +1-418-656-4747; fax: +1-418-656-4762. E-mail address: [email protected] (M. Laviolette).
Finotto, & Denburg, 1995; Wardlaw, Brightling, Green, Woltmann, & Pavord, 2000). Transendothelial migration and emigration of eosinophils within the extracellular matrix (ECM) requires the secretion and activation of proteinases, notably the matrix metalloproteinases (MMPs) (Ohbayashi, 2002; Owen & Campbell, 1999; Sternlicht & Werb, 2001). Besides their implication in cell migration, these endopeptidases also play an important role in the normal maintenance and turnover of ECM proteins in connective tissues and basement membranes, tissue remodeling, and normal embryogenesis (Ohbayashi, 2002). In
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contrast to most soluble MMPs, which are released as zymogens and act at distant sites following activation, degradation of ECM by membrane type (MT)-MMPs occurs mostly at cell periphery (Sternlicht & Werb, 2001). Of the 19 soluble MMPs and 6 MT-MMPs (Sternlicht & Werb, 2001), at least 13 are expressed in leukocytes including MT4-MMP (Owen & Campbell, 1999; Pap et al., 2000; Pei, 1999; Puente, Pendás, Llano, Velasco, & López-Ot´ın, 1996). MT4-MMP is a glycosylphosphatidylinositol-anchored protein (Itoh et al., 1999) that shows less than 40% sequence homology with other family members (Kolkenbrock, Essers, Ulbrich, & Will, 1999). The exact physiological role of MT4-MMP is still unclear. For instance, the ability of MT4-MMP to activate proMMP-2 or to degrade gelatin is supported by some, but not all studies (English et al., 2000; Kolkenbrock et al., 1999; Wang, Johnson, Ye, & Dyer, 1999). In humans, MT4-MMP is expressed in normal brain, colon and reproductive tissues, as well as carcinomas from numerous tissues (Grant, Giambernardi, Grant, & Klebe, 1999; Puente et al., 1996). Most importantly, the high expression of MT4-MMP in leukocytes led Puente and colleagues to suggest novel roles for this proteinase in the activation of cell surface proteinases and membrane-bound precursors of growth factors and inflammatory mediators (Puente et al., 1996). This exciting hypothesis was recently supported by studies showing that MT4-MMP may act as a TNF-␣ convertase (English et al., 2000; Wang et al., 1999) and may interact with insulin-like growth factor (Winkler & Fowlkes, 2002). Moreover, the colocalization of MT4-MMP and urokinase plasminogen activator receptor (uPAR) in the same microdomains on the cell surface suggests a putative interaction between MT4-MMP and the plasminogen–plasmin system in pericellular ECM degradation (Itoh et al., 1999). It is noteworthy that the uPA/uPAR system is expressed in eosinophils (Mabilat-Pragnon et al., 1997). Eosinophils express MMP-9 and this expression is increased following treatment with TNF-␣ (Ohno et al., 1997; Schwingshackl, Duszyk, Brown, & Moqbel, 1999). Moreover, MMP-9 plays an important role for ligand-induced eosinophil migration through ECM components (Guilbert et al., 1999; Okada, Kita, George, Gleich, & Leiferman, 1997). However, in these experiments, inhibition of MMP-9 and uPAR did not completely block eosinophil transmigration,
suggesting the implication of other proteinases. Since the expression of MT4-MMP was observed in human blood leukocytes (Puente et al., 1996) and in monocytes/macrophages (English et al., 2000), the objective of this study was to investigate the expression of MT4MMP in purified human blood eosinophils and its modulation by TNF-␣. In the present study, we show that eosinophils constitutively express MT4-MMP at the mRNA and protein levels, and that this expression is increased upon treatment with TNF-␣. 2. Materials and methods 2.1. Peripheral blood leukocyte isolation and cell culture Peripheral blood leukocytes were obtained from stable mild atopic asthmatics as previously described (Guilbert et al., 1999). Nasal polyp sections were obtained from surgery samples from a subject presenting asthma and chronic sinusitis. Approval for the study was obtained from the local ethics committee and all subjects signed an informed-consent form. Blood eosinophils were purified by negative selection with immunomagnetic bead conjugated with monoclonal anti-CD16, -CD3 and -CD19 antibodies for 20 min at 4 ◦ C and passed through a magnetic cell sorter (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The neutrophils were collected by column backwash. The mononuclear cells isolated on Ficoll–Paque gradient were collected and incubated in RPMI 1640 culture medium, supplemented with 10% FBS, in 75 cm2 culture flask for 1 h in a 5% CO2 atmosphere at 37 ◦ C to allow the adhesion of monocytes. Adherent cells were harvested by trypsinization, washed and re-suspended in RPMI 1640. Non-adherent cells were filtered through a nylon column to enrich them in T lymphocytes. Viability of each cell preparation, determined by trypan blue exclusion, was always ≥99%. Purity of leukocyte preparations performed on Diff-Quik® stained cytospin preparations was ≥99% for T lymphocytes, monocytes and eosinophils, and ≥95% for neutrophils. Eosinophil preparations were only contaminated by neutrophils while neutrophil preparations were mainly contaminated by eosinophils. The breast carcinoma MCF-7 (ATCC HTB-22) cell line, kindly provided by Dr. Bernard Tˆetu
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(Hˆotel-Dieu de Québec, Quebec City, Que.), was grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin in a 5% CO2 atmosphere at 37 ◦ C. When the cultures reached ∼85% confluence, cells were harvested, suspended in fresh culture medium, and seeded at a density of 106 cells/ml. 2.2. TNF-α stimulation of purified blood eosinophils Eosinophils (106 cells/ml) were resuspended in RPMI 1640 culture medium, supplemented with 10% FBS and 1% penicillin/streptomycin, and stimulated at 37 ◦ C in a 5% CO2 atmosphere for 0, 4, 8, 18 and 24 h with or without TNF-␣ (100 ng/ml) (PeproTech, Rocky Hill, NJ), IL-5 (10 ng/ml) (PeproTech) or both cytokines. 2.3. RT-PCR amplification of MT4-MMP mRNA Total RNA was isolated with a RNeasy kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions. Extracts were treated with RNase-free DNase (Promega Corp., Madison, WI) and quantified by fluorescence using the RiboGreen® reagent (Molecular Probes Inc., Eugene, OR). Fifty nanograms of RNA was reverse transcribed using random hexameres (Amersham Biosciences Corp., Piscataway, NJ) and Sensiscript reverse transcriptase (Qiagen). cDNA for MT4-MMP was amplified by PCR with primers for MT4-MMP whose flanks aligned from nucleotides 677 to 993 (forward primer: 5 -CTGGAGCGACATTGCGCCCCT-3 ; reverse primer: 5 -GGCCCTGGTAGTACGGCCGCA-3 ) (Puente et al., 1996). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard. The PCR reactions were carried out on a DNA Peltier Thermal Cycler 200 (MJ Research, Watertown, MA) for 30 cycles with the following settings: denaturation at 94 ◦ C for 1 min (2 min for the first cycle), annealing at 60 ◦ C for 30 s, and extension at 72 ◦ C for 1 min (10 min for the last cycle). After amplification, the PCR products were resolved by electrophoresis on ethidium bromide (EtBr)-stained 2.0% agarose gels and were visualized with ultraviolet illumination. Amplified fragment sizes for MT4-MMP and GAPDH were visualized at 317 and 220 bp, respectively.
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2.4. Analysis of the MT4-MMP protein For the analysis of MT4-MMP by Western blot, cell incubations were stopped by rapid centrifugation and cell pellet were resuspended in 1 volume of lysis buffer (10 mM Tris–HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 , 1 mM EDTA, 10 g/ml leupeptin, 10 g/ml aprotinin, 1 mM phenylmethanesulfonyl fluoride (PMSF), and 1 mM diisopropyl fluorophosphate (DFP), mixed with an equal volume of 2× Laemmli sample buffer, and immediately heated (5 min, 95 ◦ C). Samples containing the equivalent of 106 eosinophils or 5 × 105 MCF-7 cells were separated on 10% polyacrylamide gels, and then transferred onto PVDF membranes. Membranes were blocked in PBS containing 0.5% Tween 20 and 5% skim milk powder. Antigenic bands were detected with a rabbit polyclonal anti-human MT4-MMP antibody (1:1000; Serotec, Oxford, UK) followed by a 1:5000 dilution of a horseradish peroxidase-conjugated goat anti-rabbit antibody (Amersham Biosciences). The blots were developed using enhanced chemiluminescence. For the analysis of MT4-MMP by immunocytochemistry, centrifuged cells were fixed in 4% paraformaldehyde for 20 min at 4 ◦ C, washed with HBSS, and then embedded in paraffin using the Cytoblock system (Thermo Shandon, Pittsburgh, PA) according to manufacturer’s instructions. Sections were immunostained as previously reported (Minshall et al., 2000) with the anti-MT4-MMP antibody. Irrelevant isotypic antibody was used as negative control. The percentage of positive cells was determined by counting 500 cells per slide in a blind manner using an Eclipse E-600 Nikon microscope. Immunohistochemistry for MT4-MMP and major basic protein (MBP) (BD Biosciences, San Jose, CA) was performed on paraffin embedded sections of a nasal polyp following the same procedure. 3. Results 3.1. Expression of MT4-MMP mRNA and its modulation by TNF-α Immediately after the isolation of each purified cell preparation, total RNA was extracted. Semiquantitative RT-PCR was performed to determine
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Fig. 1. (A) Eosinophils, lymphocytes and monocytes, but not neutrophils, express MT4-MMP mRNA. RT-PCR analysis of MT4-MMP mRNA expression by unstimulated human peripheral blood leukocytes: lane 1, DNA ladder; lanes 2–3, MCF-7 cells; lanes 4–5, eosinophils; lanes 6–7, lymphocytes; lanes 8–9, monocytes; lanes 10–11, neutrophils. (B) TNF-␣ time-dependently upregulates MT4-MMP mRNA expression. RT-PCR analysis of MT4-MMP mRNA expression by purified blood eosinophils stimulated with TNF-␣ (100 ng/ml) for different periods of time. All conditions were done in presence of IL-5 (10 ng/ml) to ensure cell viability. GAPDH was done as a control for each RT-PCR reaction and loading. This representative set of results was repeated with eosinophils obtained from two additional subjects.
whether the different leukocyte populations contained the mRNA transcript for MT4-MMP. As shown in Fig. 1A, a 317 bp band, specific for MT4-MMP, was found in purified eosinophils, lymphocytes, monocytes, and MCF-7 cells that express MT4-MMP (Grant et al., 1999). In contrast, no amplification of the MT4-MMP mRNA was observed with the total RNA extracts obtained from neutrophil cell preparations. This pattern of mRNA expression was consistently found in purified leukocytes from three subjects. Given that TNF-␣ induces MMPs and MT-MMPs (Han, Tuan, Wu, Hughes, & Garner, 2001; Leber & Balkwill, 1998; Schwingshackl et al., 1999), we investigated herein the impact of this cytokine on MT4-MMP gene expression in purified blood eosinophils. As expected, treatment of eosinophils with TNF-␣, in presence of 10 ng/ml IL-5 to ensure adequate cell viability, increased the expression of MT4-MMP mRNA (Fig. 1B). This effect was maximal after 8 h and returned to baseline at 18 h. 3.2. MT4-MMP protein expression in eosinophils and its modulation by TNF-α The MT4-MMP protein was detected by Western blot on freshly purified blood eosinophils and on
MCF-7 cells (Fig. 2A). We were unable to detect any MT4-MMP protein using the same approach in purified blood neutrophils, negative for MT4-MMP mRNA (data not shown). These experiments were performed in three subjects. The incubation of eosinophils with TNF-␣, in the presence or not of IL-5, resulted in an important increase of MT4-MMP protein expression (Fig. 2B), confirming the ability of TNF-␣ to modulate the levels of MT4-MMP in eosinophils. As measured by densitometry, MT4-MMP expression increased by 3.2-fold in presence of TNF-␣ (n = 5, P = 0.01, randomized block design followed by a Tukey’s procedure). The combination of TNF-␣ and IL-5 did not further increase MT4-MMP expression. In these experiments, an incubation time of 18 h was chosen based on the maximal increase in MT4-MMP mRNA obtained at 8 h. Immunocytochemistry performed on eosinophils incubated 18 h with IL-5 further confirmed the presence of MT4-MMP in eosinophils (Fig. 2C). Mean MT4-MMP-positive cell count was 65.7 ± 7.5% (n = 3). Fig. 2D shows the absence of staining with the isotypic control antibody. Immunohistochemistry performed on nasal polyp sections showed MT4-MMP positive eosinophils demonstrating the expression of this MMP in tissue eosinophils (Fig. 3A). Eosinophils were identified by morphology and MPB positivity (Fig. 3B) and the isotypic control showed no staining (Fig. 3C).
4. Discussion In the present study, we showed by RT-PCR that purified human blood eosinophils express mRNA for MT4-MMP. In contrast, amplification of the MT4-MMP mRNA was unsuccessful in purified neutrophils strongly suggesting that these granulocytes do not express MT4-MMP. Since the only contaminant of our eosinophil preparations were neutrophils, this negative result gives us strong evidence that the presence of the MT4-MMP mRNA in human eosinophil is specific. In perfect agreement with the results obtained at the mRNA level, we also demonstrated by two different approaches, namely Western blot and immunocytochemistry, that human eosinophils constitutively express the MT4-MMP protein. Monocytes and lymphocytes also express MT4-MMP mRNA but we did not evaluate the presence of the protein in
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Fig. 2. (A) MT4-MMP protein expression in purified blood eosinophils. Representative (n = 4) example of MT4-MMP protein expression measured by Western blotting (10% polyacrylamide gel) in unstimulated eosinophils (lane 2) and MCF-7 cells (lane 1) used as positive control. MT4-MMP has a molecular weight of 70 kDa. (B) TNF-␣ increases eosinophil expression of MT4-MMP (a is different from b, n = 5, P = 0.01, randomized block design followed by a Tukey’s procedure). (C and D) MT4-MMP protein expression is further confirmed by immunocytochemistry. Eosinophils incubated overnight with IL-5 and immunostained for MT4-MMP expressed MT4-MMP (C). Isotypic control shows no staining (D).
Fig. 3. (A) Immunolocalization of MT4-MMP in nasal polyp eosinophils. (B) MBP staining was used to further confirm the identity of the eosinophils. (C) Isotypic control shows no staining.
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these cells. Furthermore, MT4-MMP expression was also demonstrated in tissue eosinophils of a nasal polyp, a chronic condition in which eosinophils are the most prevalent inflammatory cell type (Jordana et al., 1995). TNF-␣, a pivotal mediator of inflammation produced by numerous inflammatory cells (Tracey & Cerami, 1994), increases bronchial hyperresponsiveness and induces the recruitment of neutrophils and eosinophils in the lung (Thomas, 2001). Furthermore, TNF-␣ markedly upregulates the production of MMP-9 in human monocytes (Leber & Balkwill, 1998) and eosinophils (Schwingshackl et al., 1999). Importantly, we found that TNF-␣ upregulates the eosinophil MT4-MMP mRNA expression in a time-dependent manner. Western blotting confirmed this result at the protein level. The TNF-␣-induced gene expression pattern of MMP-9 and MT4-MMP by eosinophils is quite different. Schwingshacki et al. observed that stimulation of eosinophils with TNF-␣ induce an increase in MMP-9 mRNA expression at 24 h (Schwingshackl et al., 1999). In contrast, in our experimental model, we have demonstrated herein that the peak increase for MT4-MMP mRNA level occurs 8 h following the exposure of eosinophils to TNF-␣. The earlier up-regulation of MT4-MMP mRNA and protein may suggest a critical role for this MMP in the early steps leading to the migration of peripheral blood leukocytes to the tissue. We previously demonstrated that the migration of eosinophils through a basement membrane requires the action of several proteinases such as MMP-9 and the plasminogen–plasmin system (Guilbert et al., 1999). However, we were unable to completely block the transmigration of the cells, suggesting that other mechanisms and/or proteinases were implicated in this process. The up-regulation of MT4-MMP by TNF-␣ (this study) and the activation of TNF-␣ by MT4-MMP (English et al., 2000; Wang et al., 1999) suggest an important cross-talking between MT4MMP and TNF-␣ in activated and transmigrating eosinophils, which definitely deserves further studies. uPAR is found in close association with 2 integrins on leukocytes and modulates integrin-mediated binding to ECM proteins (Sitrin et al., 1996). Since MT4-MMP has been shown to colocalize with uPAR on transfected CHO-K1 cells (Itoh et al., 1999), it may also be involved in the regulation of 2 integrin
functions. Moreover, although the proteolytic capacity of MT4-MMP is not well defined yet (Ohbayashi, 2002), this membrane-anchored proteinase may promote pericellular proteolysis, preserving the overall structural integrity of the ECM. MT4-MMP may also interact with other proteins to orientated and spatially limited ECM proteolysis occurring during cell migration into tissue. In conclusion, the ability of eosinophils to express MT4-MMP may indicate a role for this proteinase in chronic diseases such as asthma and nasal polyposis. It may enhance signaling for firm leukocyte adhesion and migration to inflammatory sites and promote tissue proteolysis and remodeling.
Acknowledgements This work was supported by the Canadian Institutes of Health Research. The authors thank Luce Trépanier for her invaluable help in recruiting and evaluating the subjects for the study, and Joanne Milot and Francine Deschesnes for blood sampling. We also thank Nathalie Pagé for judicious advice in cell preparation and analysis. References English, W. R., Puente, X. S., Freije, J. M. P., Knäuper, V., Amour, A., Merryweather, A., López-Ot´ın, C., & Murphy, G. (2000). Membrane type 4 matrix metalloproteinase (MMP17) has tumor necrosis factor-␣ convertase activity but does not activate pro-MMP2. The Journal of Biological Chemistry, 275, 14046–14055. Grant, G. M., Giambernardi, T. A., Grant, A. M., & Klebe, R. J. (1999). Overview of expression of matrix metalloproteinases (MMP-17, MMP-18, and MMP-20) in cultured human cells. Matrix Biology, 18, 145–148. Guilbert, M., Ferland, C., Bossé, M., Flamand, N., Lavigne, S., & Laviolette, M. (1999). 5-Oxo-6,8,11,14-eicosatetraenoic acid induces important eosinophil transmigration through basement membrane components: Comparison of normal and asthmatic eosinophils. American Journal of Respiratory Cell and Molecular Biology, 21, 97–104. Han, Y.-P., Tuan, T.-L., Wu, H., Hughes, M., & Garner, W. L. (2001). TNF-␣ stimulates activation of pro-MMP2 in human skin through NF-B mediated induction of MT1-MMP. Journal of Cell Science, 114, 131–139. Itoh, Y., Kajita, M., Kinoh, H., Mori, H., Okada, A., & Seiki, M. (1999). Membrane type 4 matrix metalloproteinase (MT4-MMP, MMP-17) is a glycosylphosphatidylinositol-
M.-C. Gauthier et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 1667–1673 anchored proteinase. The Journal of Biological Chemistry, 274, 34260–34266. Jordana, M., Dolovich, J., Ohno, I., Finotto, S., & Denburg, J. (1995). Nasal polyposis: A model for chronic inflammation. In W. W. Busse, & S. T. Holgate (Eds.), Asthma and rhinitis (pp. 156–164). Boston: Blackwell Scientific Publications. Kolkenbrock, H., Essers, L., Ulbrich, N., & Will, H. (1999). Biochemical characterization of the catalytic domain of membrane-type 4 matrix metalloproteinase. Biological Chemistry, 380, 1103–1108. Leber, T. M., & Balkwill, F. R. (1998). Regulation of monocyte MMP-9 production by TNF-alpha and a tumour-derived soluble factor (MMPSF). British Journal of Cancer, 78, 724– 732. Mabilat-Pragnon, C., Janin, A., Michel, L., Thomaidis, A., Legrand, Y., Soria, C., & Lu, H. (1997). Urokinase localization and activity in isolated eosinophils. Thrombosis Research, 88, 373–379. Minshall, E., Chakir, J., Laviolette, M., Molet, S., Zhu, Z., Olivenstein, R., Elias, J. A., & Hamid, Q. (2000). IL-11 expression is increased in severe asthma: Association with epithelial cells and eosinophils. Journal of Allergy and Clinical Immunology, 105, 232–238. Ohbayashi, H. (2002). Matrix metalloproteinases in lung diseases, Current Protein. Current Protein & Peptide Science, 3, 409– 421. Ohno, I., Ohtani, H., Nitta, Y., Suzuki, J., Hoshi, H., Honma, M., Isoyama, S., Tanno, Y., Tamura, G., Yamauchi, K., Nagura, H., & Shirato, K. (1997). Eosinophils as a source of matrix metalloproteinase-9 in asthmatic airway inflammation. American Journal of Respiratory Cell and Molecular Biology, 16, 212–219. Okada, S., Kita, H., George, T. J., Gleich, G. J., & Leiferman, K. M. (1997). Migration of eosinophils through basement membrane components in vitro: Role of matrix metalloproteinase-9. American Journal of Respiratory Cell and Molecular Biology, 17, 519–528. Owen, C. A., & Campbell, E. J. (1999). The cell biology of leukocyte-mediated proteolysis. Journal of Leukocyte Biology, 65, 137–150.
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Pap, T., Shigeyama, Y., Kuchen, S., Fernihough, J. K., Simmen, B., Gay, R. E., Billingham, M., & Gay, S. (2000). Differential expression pattern of membrane-type matrix metalloproteinases in rheumatoid arthritis. Arthritis and Rheumatism, 43, 1226– 1232. Pei, D. (1999). Leukolysin/MMP25/MT6-MMP: A novel matrix metalloproteinase specifically expressed in the leukocyte lineage. Cell Research, 9, 291–303. Puente, X. S., Pendás, A. M., Llano, E., Velasco, G., & López-Ot´ın, C. (1996). Molecular cloning of a novel membrane-type matrix metalloproteinase from a human breast carcinoma. Cancer Research, 56, 944–949. Schwingshackl, A., Duszyk, M., Brown, N., & Moqbel, R. (1999). Human eosinophils release matrix metalloproteinase-9 on stimulation with TNF-␣. Journal of Allergy and Clinical Immunology, 104, 983–990. Sitrin, R. G., Todd III, R. F., Petty, H. R., Brock, T. G., Shollenberger, S. B., Albrecht, E., & Gyetko, M. R. (1996). The urokinase receptor (CD87) facilitates CD11b/CD18-mediated adhesion of human monocytes. Journal of Clinical Investigation, 97, 1942–1951. Sternlicht, M. D., & Werb, Z. (2001). How matrix metalloproteinases regulate cell behavior. Annual Review of Cell and Developmental Biology, 17, 463–516. Thomas, P. S. (2001). Tumour necrosis factor-␣: The role of this multifunctional cytokine in asthma. Immunology and Cell Biology, 79, 132–140. Tracey, K. J., & Cerami, A. (1994). Tumor necrosis factor: A pleiotropic cytokine and therapeutic target. Annual Review of Medicine, 45, 491–503. Wang, Y., Johnson, A. R., Ye, Q.-Z., & Dyer, R. D. (1999). Catalytic activities and substrate specificity of the human membrane type 4 matrix metalloproteinase catalytic domain. Journal of Biological Chemistry, 274, 33043–33049. Wardlaw, A. J., Brightling, C., Green, R., Woltmann, G., & Pavord, I. (2000). Eosinophils in asthma and other allergic diseases. British Medical Bulletin, 56, 985–1003. Winkler, M. K., & Fowlkes, J. L. (2002). Metalloproteinase and growth factor interactions: Do they play a role in pulmonary fibrosis? American Journal of Physiology, 283, L1–L11.
Expression of membrane type-4 matrix metalloproteinase (metalloproteinase-17) by human eosinophils Marie-Christine Gauthier, Christine Racine, Claudine Ferland, Nicolas Flamand, Jamila Chakir, Guy M. Tremblay, Michel Laviolette∗ Unité de Recherche en Pneumologie, Centre de Recherche de l’Hˆopital Laval, 2725, Chemin Ste-Foy, Institut Universitaire de Cardiologie et de Pneumologie de l’Université Laval, Sainte-Foy, Que., Canada G1V 4G5 Received 24 January 2003; received in revised form 20 March 2003; accepted 25 March 2003
Abstract Circulating eosinophils need proteinases to mediate a spatially limited and orientated digestion of the extracellular matrix and to migrate into tissue. Moreover, proteinases are likely involved in tissue remodeling, a crucial feature of chronic diseases including asthma. Eosinophils express matrix metalloproteinase (MMP)-9, which is increased upon stimulation with TNF-␣. Other MMPs, the membrane type (MT)-MMPs, likely play a major role in cell invasion and tissue remodeling. MT4-MMP was identified in peripheral blood leukocyte preparations, but it is not known whether eosinophils express MT4-MMP. We investigated the expression of MT4-MMP and its modulation by TNF-␣ in purified human blood eosinophils. The constitutive expression of MT4-MMP mRNA was detected by RT-PCR in unstimulated eosinophils, lymphocytes, and monocytes, but not neutrophils. Stimulation of eosinophils with TNF-␣ increased MT4-MMP mRNA expression. This effect appeared at 4 h and reached a maximum at 8 h of incubation. MT4-MMP protein was detected in freshly isolated blood eosinophils by Western blotting and immunocytochemistry. TNF-␣ increased expression of the MT4-MMP protein. MT4-MMP protein was also detected in nasal polyp eosinophils by immunohistochemistry. In conclusion, eosinophils constitutively express MT4-MMP, which is increased upon stimulation with TNF-␣. Consequently, MT4-MMP may be directly involved in the degradation of extracellular matrix components and/or modulate the activity of other proteins implicated in eosinophil migration and tissue remodeling. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Expression; Membrane type-4 matrix metalloproteinase; Human eosinophils
1. Introduction Tissue accumulation of eosinophils is a prominent feature of chronic conditions such as allergy, asthma and nasal polyposis (Jordana, Dolovich, Ohno, Abbrevitions: ECM, extracellular matrix; MBP, major basic protein; MMP, matrix metalloproteinase; MT-MMP, membrane type-matrix metalloproteinase; uPAR, urokinase plasminogen activator receptor ∗ Corresponding author. Tel.: +1-418-656-4747; fax: +1-418-656-4762. E-mail address: [email protected] (M. Laviolette).
Finotto, & Denburg, 1995; Wardlaw, Brightling, Green, Woltmann, & Pavord, 2000). Transendothelial migration and emigration of eosinophils within the extracellular matrix (ECM) requires the secretion and activation of proteinases, notably the matrix metalloproteinases (MMPs) (Ohbayashi, 2002; Owen & Campbell, 1999; Sternlicht & Werb, 2001). Besides their implication in cell migration, these endopeptidases also play an important role in the normal maintenance and turnover of ECM proteins in connective tissues and basement membranes, tissue remodeling, and normal embryogenesis (Ohbayashi, 2002). In
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contrast to most soluble MMPs, which are released as zymogens and act at distant sites following activation, degradation of ECM by membrane type (MT)-MMPs occurs mostly at cell periphery (Sternlicht & Werb, 2001). Of the 19 soluble MMPs and 6 MT-MMPs (Sternlicht & Werb, 2001), at least 13 are expressed in leukocytes including MT4-MMP (Owen & Campbell, 1999; Pap et al., 2000; Pei, 1999; Puente, Pendás, Llano, Velasco, & López-Ot´ın, 1996). MT4-MMP is a glycosylphosphatidylinositol-anchored protein (Itoh et al., 1999) that shows less than 40% sequence homology with other family members (Kolkenbrock, Essers, Ulbrich, & Will, 1999). The exact physiological role of MT4-MMP is still unclear. For instance, the ability of MT4-MMP to activate proMMP-2 or to degrade gelatin is supported by some, but not all studies (English et al., 2000; Kolkenbrock et al., 1999; Wang, Johnson, Ye, & Dyer, 1999). In humans, MT4-MMP is expressed in normal brain, colon and reproductive tissues, as well as carcinomas from numerous tissues (Grant, Giambernardi, Grant, & Klebe, 1999; Puente et al., 1996). Most importantly, the high expression of MT4-MMP in leukocytes led Puente and colleagues to suggest novel roles for this proteinase in the activation of cell surface proteinases and membrane-bound precursors of growth factors and inflammatory mediators (Puente et al., 1996). This exciting hypothesis was recently supported by studies showing that MT4-MMP may act as a TNF-␣ convertase (English et al., 2000; Wang et al., 1999) and may interact with insulin-like growth factor (Winkler & Fowlkes, 2002). Moreover, the colocalization of MT4-MMP and urokinase plasminogen activator receptor (uPAR) in the same microdomains on the cell surface suggests a putative interaction between MT4-MMP and the plasminogen–plasmin system in pericellular ECM degradation (Itoh et al., 1999). It is noteworthy that the uPA/uPAR system is expressed in eosinophils (Mabilat-Pragnon et al., 1997). Eosinophils express MMP-9 and this expression is increased following treatment with TNF-␣ (Ohno et al., 1997; Schwingshackl, Duszyk, Brown, & Moqbel, 1999). Moreover, MMP-9 plays an important role for ligand-induced eosinophil migration through ECM components (Guilbert et al., 1999; Okada, Kita, George, Gleich, & Leiferman, 1997). However, in these experiments, inhibition of MMP-9 and uPAR did not completely block eosinophil transmigration,
suggesting the implication of other proteinases. Since the expression of MT4-MMP was observed in human blood leukocytes (Puente et al., 1996) and in monocytes/macrophages (English et al., 2000), the objective of this study was to investigate the expression of MT4MMP in purified human blood eosinophils and its modulation by TNF-␣. In the present study, we show that eosinophils constitutively express MT4-MMP at the mRNA and protein levels, and that this expression is increased upon treatment with TNF-␣. 2. Materials and methods 2.1. Peripheral blood leukocyte isolation and cell culture Peripheral blood leukocytes were obtained from stable mild atopic asthmatics as previously described (Guilbert et al., 1999). Nasal polyp sections were obtained from surgery samples from a subject presenting asthma and chronic sinusitis. Approval for the study was obtained from the local ethics committee and all subjects signed an informed-consent form. Blood eosinophils were purified by negative selection with immunomagnetic bead conjugated with monoclonal anti-CD16, -CD3 and -CD19 antibodies for 20 min at 4 ◦ C and passed through a magnetic cell sorter (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The neutrophils were collected by column backwash. The mononuclear cells isolated on Ficoll–Paque gradient were collected and incubated in RPMI 1640 culture medium, supplemented with 10% FBS, in 75 cm2 culture flask for 1 h in a 5% CO2 atmosphere at 37 ◦ C to allow the adhesion of monocytes. Adherent cells were harvested by trypsinization, washed and re-suspended in RPMI 1640. Non-adherent cells were filtered through a nylon column to enrich them in T lymphocytes. Viability of each cell preparation, determined by trypan blue exclusion, was always ≥99%. Purity of leukocyte preparations performed on Diff-Quik® stained cytospin preparations was ≥99% for T lymphocytes, monocytes and eosinophils, and ≥95% for neutrophils. Eosinophil preparations were only contaminated by neutrophils while neutrophil preparations were mainly contaminated by eosinophils. The breast carcinoma MCF-7 (ATCC HTB-22) cell line, kindly provided by Dr. Bernard Tˆetu
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(Hˆotel-Dieu de Québec, Quebec City, Que.), was grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin in a 5% CO2 atmosphere at 37 ◦ C. When the cultures reached ∼85% confluence, cells were harvested, suspended in fresh culture medium, and seeded at a density of 106 cells/ml. 2.2. TNF-α stimulation of purified blood eosinophils Eosinophils (106 cells/ml) were resuspended in RPMI 1640 culture medium, supplemented with 10% FBS and 1% penicillin/streptomycin, and stimulated at 37 ◦ C in a 5% CO2 atmosphere for 0, 4, 8, 18 and 24 h with or without TNF-␣ (100 ng/ml) (PeproTech, Rocky Hill, NJ), IL-5 (10 ng/ml) (PeproTech) or both cytokines. 2.3. RT-PCR amplification of MT4-MMP mRNA Total RNA was isolated with a RNeasy kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions. Extracts were treated with RNase-free DNase (Promega Corp., Madison, WI) and quantified by fluorescence using the RiboGreen® reagent (Molecular Probes Inc., Eugene, OR). Fifty nanograms of RNA was reverse transcribed using random hexameres (Amersham Biosciences Corp., Piscataway, NJ) and Sensiscript reverse transcriptase (Qiagen). cDNA for MT4-MMP was amplified by PCR with primers for MT4-MMP whose flanks aligned from nucleotides 677 to 993 (forward primer: 5 -CTGGAGCGACATTGCGCCCCT-3 ; reverse primer: 5 -GGCCCTGGTAGTACGGCCGCA-3 ) (Puente et al., 1996). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard. The PCR reactions were carried out on a DNA Peltier Thermal Cycler 200 (MJ Research, Watertown, MA) for 30 cycles with the following settings: denaturation at 94 ◦ C for 1 min (2 min for the first cycle), annealing at 60 ◦ C for 30 s, and extension at 72 ◦ C for 1 min (10 min for the last cycle). After amplification, the PCR products were resolved by electrophoresis on ethidium bromide (EtBr)-stained 2.0% agarose gels and were visualized with ultraviolet illumination. Amplified fragment sizes for MT4-MMP and GAPDH were visualized at 317 and 220 bp, respectively.
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2.4. Analysis of the MT4-MMP protein For the analysis of MT4-MMP by Western blot, cell incubations were stopped by rapid centrifugation and cell pellet were resuspended in 1 volume of lysis buffer (10 mM Tris–HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 , 1 mM EDTA, 10 g/ml leupeptin, 10 g/ml aprotinin, 1 mM phenylmethanesulfonyl fluoride (PMSF), and 1 mM diisopropyl fluorophosphate (DFP), mixed with an equal volume of 2× Laemmli sample buffer, and immediately heated (5 min, 95 ◦ C). Samples containing the equivalent of 106 eosinophils or 5 × 105 MCF-7 cells were separated on 10% polyacrylamide gels, and then transferred onto PVDF membranes. Membranes were blocked in PBS containing 0.5% Tween 20 and 5% skim milk powder. Antigenic bands were detected with a rabbit polyclonal anti-human MT4-MMP antibody (1:1000; Serotec, Oxford, UK) followed by a 1:5000 dilution of a horseradish peroxidase-conjugated goat anti-rabbit antibody (Amersham Biosciences). The blots were developed using enhanced chemiluminescence. For the analysis of MT4-MMP by immunocytochemistry, centrifuged cells were fixed in 4% paraformaldehyde for 20 min at 4 ◦ C, washed with HBSS, and then embedded in paraffin using the Cytoblock system (Thermo Shandon, Pittsburgh, PA) according to manufacturer’s instructions. Sections were immunostained as previously reported (Minshall et al., 2000) with the anti-MT4-MMP antibody. Irrelevant isotypic antibody was used as negative control. The percentage of positive cells was determined by counting 500 cells per slide in a blind manner using an Eclipse E-600 Nikon microscope. Immunohistochemistry for MT4-MMP and major basic protein (MBP) (BD Biosciences, San Jose, CA) was performed on paraffin embedded sections of a nasal polyp following the same procedure. 3. Results 3.1. Expression of MT4-MMP mRNA and its modulation by TNF-α Immediately after the isolation of each purified cell preparation, total RNA was extracted. Semiquantitative RT-PCR was performed to determine
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Fig. 1. (A) Eosinophils, lymphocytes and monocytes, but not neutrophils, express MT4-MMP mRNA. RT-PCR analysis of MT4-MMP mRNA expression by unstimulated human peripheral blood leukocytes: lane 1, DNA ladder; lanes 2–3, MCF-7 cells; lanes 4–5, eosinophils; lanes 6–7, lymphocytes; lanes 8–9, monocytes; lanes 10–11, neutrophils. (B) TNF-␣ time-dependently upregulates MT4-MMP mRNA expression. RT-PCR analysis of MT4-MMP mRNA expression by purified blood eosinophils stimulated with TNF-␣ (100 ng/ml) for different periods of time. All conditions were done in presence of IL-5 (10 ng/ml) to ensure cell viability. GAPDH was done as a control for each RT-PCR reaction and loading. This representative set of results was repeated with eosinophils obtained from two additional subjects.
whether the different leukocyte populations contained the mRNA transcript for MT4-MMP. As shown in Fig. 1A, a 317 bp band, specific for MT4-MMP, was found in purified eosinophils, lymphocytes, monocytes, and MCF-7 cells that express MT4-MMP (Grant et al., 1999). In contrast, no amplification of the MT4-MMP mRNA was observed with the total RNA extracts obtained from neutrophil cell preparations. This pattern of mRNA expression was consistently found in purified leukocytes from three subjects. Given that TNF-␣ induces MMPs and MT-MMPs (Han, Tuan, Wu, Hughes, & Garner, 2001; Leber & Balkwill, 1998; Schwingshackl et al., 1999), we investigated herein the impact of this cytokine on MT4-MMP gene expression in purified blood eosinophils. As expected, treatment of eosinophils with TNF-␣, in presence of 10 ng/ml IL-5 to ensure adequate cell viability, increased the expression of MT4-MMP mRNA (Fig. 1B). This effect was maximal after 8 h and returned to baseline at 18 h. 3.2. MT4-MMP protein expression in eosinophils and its modulation by TNF-α The MT4-MMP protein was detected by Western blot on freshly purified blood eosinophils and on
MCF-7 cells (Fig. 2A). We were unable to detect any MT4-MMP protein using the same approach in purified blood neutrophils, negative for MT4-MMP mRNA (data not shown). These experiments were performed in three subjects. The incubation of eosinophils with TNF-␣, in the presence or not of IL-5, resulted in an important increase of MT4-MMP protein expression (Fig. 2B), confirming the ability of TNF-␣ to modulate the levels of MT4-MMP in eosinophils. As measured by densitometry, MT4-MMP expression increased by 3.2-fold in presence of TNF-␣ (n = 5, P = 0.01, randomized block design followed by a Tukey’s procedure). The combination of TNF-␣ and IL-5 did not further increase MT4-MMP expression. In these experiments, an incubation time of 18 h was chosen based on the maximal increase in MT4-MMP mRNA obtained at 8 h. Immunocytochemistry performed on eosinophils incubated 18 h with IL-5 further confirmed the presence of MT4-MMP in eosinophils (Fig. 2C). Mean MT4-MMP-positive cell count was 65.7 ± 7.5% (n = 3). Fig. 2D shows the absence of staining with the isotypic control antibody. Immunohistochemistry performed on nasal polyp sections showed MT4-MMP positive eosinophils demonstrating the expression of this MMP in tissue eosinophils (Fig. 3A). Eosinophils were identified by morphology and MPB positivity (Fig. 3B) and the isotypic control showed no staining (Fig. 3C).
4. Discussion In the present study, we showed by RT-PCR that purified human blood eosinophils express mRNA for MT4-MMP. In contrast, amplification of the MT4-MMP mRNA was unsuccessful in purified neutrophils strongly suggesting that these granulocytes do not express MT4-MMP. Since the only contaminant of our eosinophil preparations were neutrophils, this negative result gives us strong evidence that the presence of the MT4-MMP mRNA in human eosinophil is specific. In perfect agreement with the results obtained at the mRNA level, we also demonstrated by two different approaches, namely Western blot and immunocytochemistry, that human eosinophils constitutively express the MT4-MMP protein. Monocytes and lymphocytes also express MT4-MMP mRNA but we did not evaluate the presence of the protein in
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Fig. 2. (A) MT4-MMP protein expression in purified blood eosinophils. Representative (n = 4) example of MT4-MMP protein expression measured by Western blotting (10% polyacrylamide gel) in unstimulated eosinophils (lane 2) and MCF-7 cells (lane 1) used as positive control. MT4-MMP has a molecular weight of 70 kDa. (B) TNF-␣ increases eosinophil expression of MT4-MMP (a is different from b, n = 5, P = 0.01, randomized block design followed by a Tukey’s procedure). (C and D) MT4-MMP protein expression is further confirmed by immunocytochemistry. Eosinophils incubated overnight with IL-5 and immunostained for MT4-MMP expressed MT4-MMP (C). Isotypic control shows no staining (D).
Fig. 3. (A) Immunolocalization of MT4-MMP in nasal polyp eosinophils. (B) MBP staining was used to further confirm the identity of the eosinophils. (C) Isotypic control shows no staining.
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these cells. Furthermore, MT4-MMP expression was also demonstrated in tissue eosinophils of a nasal polyp, a chronic condition in which eosinophils are the most prevalent inflammatory cell type (Jordana et al., 1995). TNF-␣, a pivotal mediator of inflammation produced by numerous inflammatory cells (Tracey & Cerami, 1994), increases bronchial hyperresponsiveness and induces the recruitment of neutrophils and eosinophils in the lung (Thomas, 2001). Furthermore, TNF-␣ markedly upregulates the production of MMP-9 in human monocytes (Leber & Balkwill, 1998) and eosinophils (Schwingshackl et al., 1999). Importantly, we found that TNF-␣ upregulates the eosinophil MT4-MMP mRNA expression in a time-dependent manner. Western blotting confirmed this result at the protein level. The TNF-␣-induced gene expression pattern of MMP-9 and MT4-MMP by eosinophils is quite different. Schwingshacki et al. observed that stimulation of eosinophils with TNF-␣ induce an increase in MMP-9 mRNA expression at 24 h (Schwingshackl et al., 1999). In contrast, in our experimental model, we have demonstrated herein that the peak increase for MT4-MMP mRNA level occurs 8 h following the exposure of eosinophils to TNF-␣. The earlier up-regulation of MT4-MMP mRNA and protein may suggest a critical role for this MMP in the early steps leading to the migration of peripheral blood leukocytes to the tissue. We previously demonstrated that the migration of eosinophils through a basement membrane requires the action of several proteinases such as MMP-9 and the plasminogen–plasmin system (Guilbert et al., 1999). However, we were unable to completely block the transmigration of the cells, suggesting that other mechanisms and/or proteinases were implicated in this process. The up-regulation of MT4-MMP by TNF-␣ (this study) and the activation of TNF-␣ by MT4-MMP (English et al., 2000; Wang et al., 1999) suggest an important cross-talking between MT4MMP and TNF-␣ in activated and transmigrating eosinophils, which definitely deserves further studies. uPAR is found in close association with 2 integrins on leukocytes and modulates integrin-mediated binding to ECM proteins (Sitrin et al., 1996). Since MT4-MMP has been shown to colocalize with uPAR on transfected CHO-K1 cells (Itoh et al., 1999), it may also be involved in the regulation of 2 integrin
functions. Moreover, although the proteolytic capacity of MT4-MMP is not well defined yet (Ohbayashi, 2002), this membrane-anchored proteinase may promote pericellular proteolysis, preserving the overall structural integrity of the ECM. MT4-MMP may also interact with other proteins to orientated and spatially limited ECM proteolysis occurring during cell migration into tissue. In conclusion, the ability of eosinophils to express MT4-MMP may indicate a role for this proteinase in chronic diseases such as asthma and nasal polyposis. It may enhance signaling for firm leukocyte adhesion and migration to inflammatory sites and promote tissue proteolysis and remodeling.
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