Degradation of Interleukin 1β by Matrix Metalloproteinases

Matrix metalloproteinases (MMPs) and interleukin 1 (IL-1) are implicated in inflammation and tissue destruction, where IL-1 is a potent stimulator of connective tissue cells to produce the extracellular matrix-degrading MMPs. Here, we report that IL-1β, but not IL-1α, is degraded by MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), and MMP-9 (gelatinase B). This degradation was effectively blocked by tissue inhibitor of metalloproteinases (TIMP)-1. When IL-1β was treated with MMPs it lost the ability to enhance the synthesis of prostaglandin E2 and pro-MMP-3 in human fibroblasts. The primary cleavage site of IL-1β by MMP-2 was identified at the Glu25-Leu26 bond. These results suggest that IL-1β stimulates connective tissue cells to produce MMPs, but activated MMPs in turn negatively regulate the activity of IL-1β. Matrix metalloproteinases (MMPs) and interleukin 1 (IL-1) are implicated in inflammation and tissue destruction, where IL-1 is a potent stimulator of connective tissue cells to produce the extracellular matrix-degrading MMPs. Here, we report that IL-1β, but not IL-1α, is degraded by MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), and MMP-9 (gelatinase B). This degradation was effectively blocked by tissue inhibitor of metalloproteinases (TIMP)-1. When IL-1β was treated with MMPs it lost the ability to enhance the synthesis of prostaglandin E2 and pro-MMP-3 in human fibroblasts. The primary cleavage site of IL-1β by MMP-2 was identified at the Glu25-Leu26 bond. These results suggest that IL-1β stimulates connective tissue cells to produce MMPs, but activated MMPs in turn negatively regulate the activity of IL-1β. INTRODUCTIONMatrix metalloproteinases (MMPs), 1The abbreviations used are: MMPmatrix metalloproteinaseTIMPtissue inhibitor of metalloproteinasesILinterleukinTNFαtumor necrosis factor αAPMA4-aminophenylmercuric acetatePAGEpolyacrylamide gel electrophoresisMEMEagle's minimum essential mediumHPLChigh performance liquid chromatography. also called matrixins, degrade extracellular matrix macromolecules and play important roles in many biological processes such as morphogenesis, ovulation, embryo implantation, cell migration, tissue involution, angiogenesis, and wound healing (1Woessner Jr., J.F. FASEB J. 1991; 5: 2145-2154Google Scholar, 2Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral. Biol. Med. 1993; 4: 197-250Google Scholar, 3Nagase H. Hooper N.M. Zinc Metalloproteinases in Health and Disease. Taylor & Francis, London1996Google Scholar). In excess, they participate in the destruction of the tissue associated with many connective tissue diseases such as arthritis, periodentitis, nephritis, and tissue ulcerations and with tumor cell invasion and metastasis (1Woessner Jr., J.F. FASEB J. 1991; 5: 2145-2154Google Scholar, 2Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral. Biol. Med. 1993; 4: 197-250Google Scholar, 3Nagase H. Hooper N.M. Zinc Metalloproteinases in Health and Disease. Taylor & Francis, London1996Google Scholar). The importance of matrixins in both physiological and pathological catabolism of extracellular matrix macromolecules has been emphasized because little MMP activities can be detected in normal steady state tissue, but the synthesis of many MMPs is transcriptionally regulated by inflammatory cytokines, hormones, growth factors, and cellular transformation (1Woessner Jr., J.F. FASEB J. 1991; 5: 2145-2154Google Scholar, 2Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral. Biol. Med. 1993; 4: 197-250Google Scholar, 3Nagase H. Hooper N.M. Zinc Metalloproteinases in Health and Disease. Taylor & Francis, London1996Google Scholar). For example, high levels of MMP-1 (interstitial collagenase, EC), MMP-3 (stromelysin 1, EC), and MMP-9 (gelatinase B, EC) are found in synovial tissues and fluids from patients with rheumatoid arthritis (4Arend W.P. Dayer J.M. Arthritis Rheum. 1990; 33: 305-315Google Scholar, 5Walakovits L.A. Moore V.L. Bhardwaj N. Gallick G.S. Lark M.W. Arthritis Rheum. 1992; 35: 35-42Google Scholar, 6Tetlow L.C. Lees M. Ogata Y. Nagase H. Woolley D.E. Rheumatol. Int. 1993; 13: 53-59Google Scholar). It is generally accepted that an elevated level of interleukin 1 (IL-1) is one of the key mediators that greatly enhances the biosynthesis and secretion of precursors of these MMPs (pro-MMPs) and prostaglandin E2 from mesenchymal cells at inflammatory sites (1Woessner Jr., J.F. FASEB J. 1991; 5: 2145-2154Google Scholar).IL-1 is secreted from activated macrophages and a variety of other cell types and elicits many other biological responses such as thymocyte proliferation, fever production, wound healing, and tissue resorption (see 7Dinarello C.A. Chem. Immunol. 1992; 51: 1-32Google Scholar for review). The promotion of wound healing and tissue degradation is considered to be in part due to the production of MMPs by cells stimulated with IL-1. The suppression of IL-1 activity is, therefore, thought to be an effective step to control inflammatory responses. In this regard, a large number of studies have focused on the regulation of IL-1 synthesis, processing of the IL-1 precursor, and the receptor antagonist (7Dinarello C.A. Chem. Immunol. 1992; 51: 1-32Google Scholar). However, little is known about the catabolism of the mature form of IL-1.In this communication, we report that MMP-1, MMP-2 (gelatinase A, EC), MMP-3, and MMP-9 secreted from the stimulated connective tissue cells degrade IL-1β but not IL-1α. Our study suggests that the MMPs induced by IL-1β negatively control the biological activities of IL-1β at the sites of inflammation and tissue injury.RESULTSDuring the course of our investigation of IL-1 precursor processing enzymes in human uterine cervical fibroblasts, we observed that the conditioned culture medium of the cells co-treated with IL-1α and TNFα contained the IL-1β-degrading activity when it was activated with APMA. As shown in Fig. 1, the conditioned culture medium degraded IL-1β at 37°C in the presence of APMA in a time-dependent manner, whereas in the absence of APMA IL-1β was intact. The degradation of IL-1β by the APMA-treated medium was completely inhibited by the presence of TIMP-1 (3 µg/ml) (Fig. 2). These results suggest that activated MMPs are responsible for degradation of IL-1β. Western blotting analysis showed that the medium of uterine cervical cells treated with IL-1α and TNFα contained pro-MMPs-1, −2, −3, and −9 (data not shown).Fig. 2TIMP-1 interferes with the degradation of IL-1β by APMA-activated MMPs. The culture medium as in Fig. 1 containing IL-1β (0.6 ng/ml) and 1 mM APMA was incubated with or without TIMP-1 (3 µg/ml) at 37°C for 24 h, and the immunoreactive IL-1β was monitored by ECL-Western blotting. Lane 1, IL-1β incubated with culture medium; lane 2, IL-1β incubated with culture medium in the presence of 1 mM APMA; and lane 3, IL-1β incubated with culture medium in the presence of 1 mM APMA and TIMP-1.View Large Image Figure ViewerDownload (PPT)To identify which MMPs in the medium degrade IL-1β, purified MMPs-1, −2, −3, and −9 were tested for their ability to digest IL-1β. As shown in Fig. 3, all four MMPs digested IL-1β in a dose-dependent manner. The lack of detection of the degradation products was due to the analytical procedure using immunoblotting. Distinct digestion products were detected by SDS-PAGE analysis followed by silver staining (see Fig. 5). By contrast, IL-1α was resistant to these four MMPs, emphasizing that MMPs selectively hydrolyze IL-1β.Fig. 3Degradation of IL-1α and β by purified MMPs-1, −2, −3, and −9. Purified pro-MMP was first activated with 1 mM APMA in 50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 10 mM CaCl2 and then incubated at 37°C for 24 h with IL-1α or IL-1β (20 ng/ml each) in the same buffer as indicated. The digestion of IL-1s was monitored by ECL-Western blotting as described under “Experimental Procedures.” Lane 1, the respective IL-1 incubated with the buffer alone.View Large Image Figure ViewerDownload (PPT)Fig. 5Cleavage of IL-1β by MMP-2. IL-1β (100 µg) was digested with MMP-2 as described under “Experimental Procedures.” Panel A, SDS-PAGE analysis of digestion products of IL-1β by MMP-2. Lane 1, IL-1β incubated with pro-MMP-2 alone; and lane 2, IL-1β incubated with pro-MMP-2 and 0.5 mM APMA. Proteins were stained with silver staining. BF, buffer front. Panel B, cleavage site of IL-1β by MMP-2. The products shown in lane 2 of panel A were separated by HPLC and subjected to the N-terminal sequence analysis as described under “Experimental Procedures.”▴, the site cleaved by MMP-2.View Large Image Figure ViewerDownload (PPT)IL-1β treated with MMP-1 or MMP-2 was tested for its biological activities on human uterine cervical fibroblasts. As shown in Fig. 4, A and B, the stimulatory activity of IL-1β on the production of prostaglandin E2 and pro-MMP-3 was reduced, and the residual activity corresponded to the amount of intact IL-1β. Pro-MMP-2 did not modulate the biological activity of IL-1β.Fig. 4Loss of biological activities of IL-1β upon hydrolysis by MMPs. Panel A, effect of MMP-1 on the IL-1β-mediated production of prostaglandin E2. IL-1β (20 ng) and 0.33 unit of MMP-1 were incubated at 37°C for 24 h in a total of 1 ml of 50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 10 mM CaCl2, and then a portion of the reaction mixture was added to confluent human uterine cervical fibroblasts in 24-multiwell plates at a final IL-1β concentration of 0.1 ng/ml, and cells were cultured for 24 h. The production of prostaglandin E2 was measured as described under “Experimental Procedures.” The amounts of prostaglandin E2 are shown as the mean ± S.D. of three wells. Lane 1, control; lane 2, vehicle only; lane 3, IL-1β; and lane 4, IL-1β treated with MMP-1. a, significantly different from lanes 1 and 2 (p < 0.01); b, significantly different from lane 3 (p < 0.01). Panel B, effect of MMP-2 on the IL-1β-mediated pro-MMP-3 production. IL-1β (2 µg) was treated with the APMA-activated MMP-2 (5.3 units) at 37°C for 24 h in a total of 160 µl of the buffer, and then the reaction mixture was added to confluent human uterine cervical cells in 12-multiwell plates at a final IL-1β concentration of 13 ng/ml. Cells were cultured for 24 h, and then pro-MMP-3 in the culture media was measured by Western blotting as described under “Experimental Procedures.” The relative amounts of pro-MMP-3 were quantified by densitometric scanning, taking the control cells as 1, and the values are shown at the top of each lane. Lane 1, control; lane 2, vehicle alone; lane 3, IL-1β; lane 4, IL-1β treated with active MMP-2; and lane 5, IL-1β treated with pro-MMP-2.View Large Image Figure ViewerDownload (PPT)To identify the site of IL-1β cleaved by MMPs, IL-1β was incubated with pro-MMP-2 in the presence of 0.5 mM APMA. Experimental conditions were chosen to obtain a partial digestion of IL-1β so that the major cleavage site can be analyzed. SDS-PAGE analysis of the digest indicated that 17.5-kDa IL-1β was cleaved into approximate 14.5- and 3-kDa fragments (Fig. 5). This sample was subjected to reverse phase HPLC, and the protein peaks were analyzed by Edman degradation. The main peak derived from MMP-2 digestion displayed the two sequences: Ala-Pro-Val-Arg (N-terminal sequence for IL-1β) and Leu-Lys-Ala-Leu. This sample was then reduced, alkylated, and separated by reverse phase HPLC, and the new N-terminal Leu-Lys-Ala-Leu sequence was confirmed. These results indicate that the primary site of IL-1β cleaved by MMP-2 is the Glu25-Leu26 bond.DISCUSSIONMatrixins secreted from cells are generally considered to degrade extracellular matrix macromolecules, but their actions on substrates other than matrix components were also reported, e.g. acute-phase reactant serum amyloid A (17Mitchell T.I. Jeffrey J.J. Palmiter R.D. Brinckerhoff C.E. Biochim. Biophys. Acta. 1993; 1156: 245-254Google Scholar), α1-proteinase inhibitors (18Desrochers P.E. Jeffrey J.J. Weiss S.J. J. Clin. Invest. 1991; 87: 2258-2265Google Scholar, 19Mast A.E. Enghild J.J. Nagase H. Suzuki K. Pizzo S.V. Salvesen G. J. Biol. Chem. 1991; 266: 15810-15816Google Scholar, 20Pei D. Majmudar G. Weiss S.J. J. Biol. Chem. 1994; 269: 25849-25855Google Scholar), α1-anti-chymotrypsin (19Mast A.E. Enghild J.J. Nagase H. Suzuki K. Pizzo S.V. Salvesen G. J. Biol. Chem. 1991; 266: 15810-15816Google Scholar), α2-macroglobulin (21Enghild J.J. Salvesen G. Nagase H. J. Biol. Chem. 1989; 264: 8779-8785Google Scholar, 22Sottrup-Jensen L. Birkedal-Hansen H. J. Biol. Chem. 1989; 264: 393-401Google Scholar), insulin-like growth factor binding protein 3 (23Fowlkes J.L. Enghild J.J. Suzuki K. Nagase H. J. Biol. Chem. 1994; 269: 25742-25746Google Scholar) and insulin-like growth factor binding protein 5 (24Thrailkill K.M. Quarles L.D. Nagase H. Suzuki K. Serra D.M. Fowlkes J.L. Endocrinology. 1995; 136: 3527-3533Google Scholar). In this report, we demonstrated that MMPs secreted from the connective tissue cells degrade IL-1β, but not IL-1α, and destroy its biological activities.The primary cleavage site of IL-1β by MMP-2 is located in the Pro-Tyr-Glu25-Leu26-Lys-Ala sequence. This stretch of residues agrees well with the sequence requirement for many MMPs, e.g. Pro at the P3 site, a Glu at the P1 subsite, Leu at the P1′, and Ala at P3′ (subsite nomenclature is after Schechter and Berger (25Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Google Scholar)) (26Netzel-Arnett S. Fields G.B. Birkedal-Hansen H. Van Wart H.E. J. Biol. Chem. 1991; 266: 6747-6755Google Scholar, 27Netzel-Arnett S. Sang Q.-X. Moore W.G.I. Navre M. Birkedal-Hansen H. Van Wart H.E. Biochemistry. 1993; 32: 6427-6432Google Scholar, 28Niedzwiecki L. Teahan J. Harrison R.K. Stein R.L. Biochemistry. 1992; 31: 12618-12623Google Scholar). The Glu25-Leu26 bond is located at the beginning of the third β-strand of IL-1β following a loop structure of Gly22-Pro-Tyr-Glu25 (29Priestle J.P. Schar H.-P. Grutter M.G. EMBO J. 1988; 7: 339-343Google Scholar). This may explain why this site is readily recognized by MMP-2 and possibly by other MMPs. The resistance of IL-1α from degradation by MMPs is most likely due to the difference in amino acid sequence between IL-1α and IL-1β as less than 30% of their amino acids are identical in sequence (30March C.J. Mosley B. Larsen A. Cerretti D.P. Braedt G. Price V. Gillis S. Henney C.S. Kronheim S.R. Grabstein K. Conlon P.J. Hopp T.P. Cosman D. Nature. 1985; 315: 641-647Google Scholar). Pierart et al. (31Pierart M.E. Najdovski T. Appelboom T.E. Deschodt-Lanckman M.M. J. Immunol. 1988; 140: 3808-3811Google Scholar) reported that neprilysin, a membrane-bound endopeptidase (neutral endopeptidase 24.11/enkephalinase, EC), hydrolyzed IL-1β, but later studies by Kimura et al. (32Kimura A. Iwamoto I. Nakagawa N. Tomioka H. Yoshida S. Immunol. Lett. 1991; 28: 109-114Google Scholar) disputed the earlier observation.The susceptibility of IL-1β to MMPs introduces a new important aspect about understanding the progression of inflammation and tissue damage. While the normal tissues contain little MMP activities, the production of pro-MMP-1, pro-MMP-3, pro-MMP-7 (matrilysin), and pro-MMP-9 is enhanced by IL-1β that is secreted from the activated macrophages and many other cell types. These matrixins are synthesized and secreted from cells as inactive precursors, but once activated, they can control the activity of IL-1β but not IL-1α. It is notable that the major type of IL-1 found in rheumatoid synovium is IL-1β (4Arend W.P. Dayer J.M. Arthritis Rheum. 1990; 33: 305-315Google Scholar). On the other hand, the biological significance of MMP-2 on IL-1β degradation is not clear at this point. However, the recent finding that the activation of pro-MMP-2 occurs on the cell surface of fibroblasts and neoplastic cells by membrane-type MMPs (33Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Google Scholar, 34Will B. Hinzmann B. Eur. J. Biochem. 1995; 231: 602-608Google Scholar, 35Takino T. Sato H. Shinagawa A. Seiki M. J. Biol. Chem. 1995; 270: 23013-23020Google Scholar) suggests that the activated MMP-2 may regulate the biological activity of IL-1β on the periphery of the target cells. Recently it has been reported that hydroxamic acid-based synthetic inhibitors of MMP prevent the release of TNFα from the endotoxin-stimulated human leukocytes in culture (36Mohler K.M. Sleath P.R. Fitzner J.N. Cerretti D.P. Alderson M. Kerwar S.S. Torrance D.S. Otten-Evans C. Greenstreet T. Weerawarna K. Kronheim S.R. Peterson M. Gerhart M. Kozlosky C.J. March C.J. Black R.A. Nature. 1994; 370: 218-220Google Scholar, 37Gearing A.J.H. Beckett P. Christodoulou M. Churchill M. Ciements J. Davidson A.H. Drummond A.H. Galloway W.A. Gilbert R. Gordon J.L. Leber T.M. Mangan M. Miller K. Nayee P. Owen K. Patel S. Thomas W. Wells G. Wood L.M. Woolley K. Nature. 1994; 370: 555-557Google Scholar, 38McGeehan G.M. Becherer J.D. Bast Jr., R.C. Boyer C.M. Champion B. Connolly K.M. Conway J.G. Furdon P. Karp S. Kidao S. MoElroy A.B. Nichols J. Pryzwansky K.M. Schoenen F. Sekut K. Truesdale A. Verghese M. Warner J. Ways J.P. Nature. 1994; 370: 558-561Google Scholar), and McGeehan et al. (38McGeehan G.M. Becherer J.D. Bast Jr., R.C. Boyer C.M. Champion B. Connolly K.M. Conway J.G. Furdon P. Karp S. Kidao S. MoElroy A.B. Nichols J. Pryzwansky K.M. Schoenen F. Sekut K. Truesdale A. Verghese M. Warner J. Ways J.P. Nature. 1994; 370: 558-561Google Scholar) noted that there was a 2-fold increase in IL-1β in the medium of the stimulated cells under the presence of a synthetic MMP inhibitor. This phenomenon may be in part attributed to the inhibition of the MMP-mediated IL-1β degradation by the peptide-hydroxamic acid inhibitor.In summary, the present study provides biochemical evidence that IL-1β is readily degraded by MMPs. This emphasizes that the catabolism of IL-1β by MMPs be into to the progression of inflammation and the connective tissue of MMP activities by synthetic inhibitors may an increase in of IL-1β. INTRODUCTIONMatrix metalloproteinases (MMPs), 1The abbreviations used are: MMPmatrix metalloproteinaseTIMPtissue inhibitor of metalloproteinasesILinterleukinTNFαtumor necrosis factor αAPMA4-aminophenylmercuric acetatePAGEpolyacrylamide gel electrophoresisMEMEagle's minimum essential mediumHPLChigh performance liquid chromatography. also called matrixins, degrade extracellular matrix macromolecules and play important roles in many biological processes such as morphogenesis, ovulation, embryo implantation, cell migration, tissue involution, angiogenesis, and wound healing (1Woessner Jr., J.F. FASEB J. 1991; 5: 2145-2154Google Scholar, 2Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral. Biol. Med. 1993; 4: 197-250Google Scholar, 3Nagase H. Hooper N.M. Zinc Metalloproteinases in Health and Disease. Taylor & Francis, London1996Google Scholar). In excess, they participate in the destruction of the tissue associated with many connective tissue diseases such as arthritis, periodentitis, nephritis, and tissue ulcerations and with tumor cell invasion and metastasis (1Woessner Jr., J.F. FASEB J. 1991; 5: 2145-2154Google Scholar, 2Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral. Biol. Med. 1993; 4: 197-250Google Scholar, 3Nagase H. Hooper N.M. Zinc Metalloproteinases in Health and Disease. Taylor & Francis, London1996Google Scholar). The importance of matrixins in both physiological and pathological catabolism of extracellular matrix macromolecules has been emphasized because little MMP activities can be detected in normal steady state tissue, but the synthesis of many MMPs is transcriptionally regulated by inflammatory cytokines, hormones, growth factors, and cellular transformation (1Woessner Jr., J.F. FASEB J. 1991; 5: 2145-2154Google Scholar, 2Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral. Biol. Med. 1993; 4: 197-250Google Scholar, 3Nagase H. Hooper N.M. Zinc Metalloproteinases in Health and Disease. Taylor & Francis, London1996Google Scholar). For example, high levels of MMP-1 (interstitial collagenase, EC), MMP-3 (stromelysin 1, EC), and MMP-9 (gelatinase B, EC) are found in synovial tissues and fluids from patients with rheumatoid arthritis (4Arend W.P. Dayer J.M. Arthritis Rheum. 1990; 33: 305-315Google Scholar, 5Walakovits L.A. Moore V.L. Bhardwaj N. Gallick G.S. Lark M.W. Arthritis Rheum. 1992; 35: 35-42Google Scholar, 6Tetlow L.C. Lees M. Ogata Y. Nagase H. Woolley D.E. Rheumatol. Int. 1993; 13: 53-59Google Scholar). It is generally accepted that an elevated level of interleukin 1 (IL-1) is one of the key mediators that greatly enhances the biosynthesis and secretion of precursors of these MMPs (pro-MMPs) and prostaglandin E2 from mesenchymal cells at inflammatory sites (1Woessner Jr., J.F. FASEB J. 1991; 5: 2145-2154Google Scholar).IL-1 is secreted from activated macrophages and a variety of other cell types and elicits many other biological responses such as thymocyte proliferation, fever production, wound healing, and tissue resorption (see 7Dinarello C.A. Chem. Immunol. 1992; 51: 1-32Google Scholar for review). The promotion of wound healing and tissue degradation is considered to be in part due to the production of MMPs by cells stimulated with IL-1. The suppression of IL-1 activity is, therefore, thought to be an effective step to control inflammatory responses. In this regard, a large number of studies have focused on the regulation of IL-1 synthesis, processing of the IL-1 precursor, and the receptor antagonist (7Dinarello C.A. Chem. Immunol. 1992; 51: 1-32Google Scholar). However, little is known about the catabolism of the mature form of IL-1.In this communication, we report that MMP-1, MMP-2 (gelatinase A, EC), MMP-3, and MMP-9 secreted from the stimulated connective tissue cells degrade IL-1β but not IL-1α. Our study suggests that the MMPs induced by IL-1β negatively control the biological activities of IL-1β at the sites of inflammation and tissue

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