Prointerleukin-18 Is Activated by Meprin β in Vitro and in Vivo in Intestinal Inflammation

Interleukin-18 (IL-18), a pro-inflammatory cytokine, is a key factor in inflammatory bowel disease (IBD). Caspase-1 activates this cytokine, but other proteases are likely involved in maturation. Because meprin metalloproteinases have been implicated in IBD, the interaction of these proteases with proIL-18 was studied. The results demonstrate that the meprin β subunit of meprins A and B cleaves proIL-18 into a smaller 17-kDa product. The cleavage is at the Asn51–Asp52 bond, a site C-terminal to caspase-1 cleavage. The cleavage occurred in vitro with a Km of 1.3 μm and in Madin-Darby canine kidney cells transfected with meprin β when proIL-18 was added to the culture medium. The product of meprin B cleavage of proIL-18 activated NF-κB in EL-4 cells, indicating that it was biologically active. To determine the physiological significance of the interactions of meprins with proIL-18, an experimental model of IBD was produced by administering dextran sulfate sodium (DSS) to wild-type and meprin β knock-out (βKO) mice, and the serum levels of active IL-18 were determined. DSS-treated meprin βKO mice had lower levels of the active cytokine in the serum compared with wild-type mice. Furthermore, in meprin αKO mice, which express meprin β but not α, active IL-18 was elevated in the serum of DSS-treated mice compared with wild-type mice, indicating that the meprin isoforms have opposing effects on the IL-18 levels in vivo. This study identifies proIL-18 as a biologically important substrate for meprin β and implicates meprins in the modulation of inflammation. Interleukin-18 (IL-18), a pro-inflammatory cytokine, is a key factor in inflammatory bowel disease (IBD). Caspase-1 activates this cytokine, but other proteases are likely involved in maturation. Because meprin metalloproteinases have been implicated in IBD, the interaction of these proteases with proIL-18 was studied. The results demonstrate that the meprin β subunit of meprins A and B cleaves proIL-18 into a smaller 17-kDa product. The cleavage is at the Asn51–Asp52 bond, a site C-terminal to caspase-1 cleavage. The cleavage occurred in vitro with a Km of 1.3 μm and in Madin-Darby canine kidney cells transfected with meprin β when proIL-18 was added to the culture medium. The product of meprin B cleavage of proIL-18 activated NF-κB in EL-4 cells, indicating that it was biologically active. To determine the physiological significance of the interactions of meprins with proIL-18, an experimental model of IBD was produced by administering dextran sulfate sodium (DSS) to wild-type and meprin β knock-out (βKO) mice, and the serum levels of active IL-18 were determined. DSS-treated meprin βKO mice had lower levels of the active cytokine in the serum compared with wild-type mice. Furthermore, in meprin αKO mice, which express meprin β but not α, active IL-18 was elevated in the serum of DSS-treated mice compared with wild-type mice, indicating that the meprin isoforms have opposing effects on the IL-18 levels in vivo. This study identifies proIL-18 as a biologically important substrate for meprin β and implicates meprins in the modulation of inflammation. Interleukin-18 (IL-18) 2The abbreviations used are: IL-18, interleukin-18; IBD, inflammatory bowel disease; DSS, dextran sulfate sodium; MS/MS, tandem mass spectrometry; MDCK, Madin-Darby canine kidney; KO, knock-out; WT, wild-type; PR-3, proteinase-3. is a pro-inflammatory cytokine that is similar in structure to IL-1β, and both cytokines play important roles in innate host defense and inflammatory processes (1Fantuzzi G. Dinarello C.A. J. Clin. Immunol. 1999; 19: 1-11Crossref PubMed Scopus (423) Google Scholar). These two cytokines are synthesized as inactive proforms that lack signal sequences and are expressed in a variety of cells, including monocytes, macrophages, and epithelial cells. Caspase-1 has been identified as one of the proteases able to generate biologically active mature forms of IL-18 and IL-1β in response to a signal such as endotoxin (1Fantuzzi G. Dinarello C.A. J. Clin. Immunol. 1999; 19: 1-11Crossref PubMed Scopus (423) Google Scholar, 2Reuter B.K. Pizarro T.T. Eur. J. Immunol. 2004; 34: 2347-2355Crossref PubMed Scopus (95) Google Scholar). The relationship between cytokine activation and exit from the cytosol is unresolved, and the cytokines in their proforms can be released from the cell in the absence of proteolytically functional caspase-1 (3Mehta V.B. Hart J. Wewers M.D. J. Biol. Chem. 2001; 276: 3820-3826Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 4Andrei C. Margiocco P. Poggi A. Lotti L.V. Torrisi M.R. Rubartelli A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9745-9750Crossref PubMed Scopus (346) Google Scholar). Meprins, tissue-specific metalloproteinases, are highly expressed in intestinal and kidney epithelial cells and in the epidermis (5Bond J.S. Matters G.L. Banerjee S. Dusheck R.E. FEBS Lett. 2005; 579: 3317-3322Crossref PubMed Scopus (76) Google Scholar, 6Becker-Pauly C. Howel M. Walker T. Vlad A. Aufenvenne K. Oji V. Lottaz D. Sterchi E.E. Debela M. Magdolen V. Traupe H. Stocker W. J. Investig. Dermatol. 2007; 127: 1115-1125Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). They are normally found extracellularly at the apical membrane of these cells or secreted into the lumen of these tissues (7Lottaz D. Hahn D. Muller S. Muller C. Sterchi E.E. Eur. J. Biochem. 1999; 259: 496-504Crossref PubMed Scopus (70) Google Scholar, 8Marchand P. Tang J. Bond J.S. J. Biol. Chem. 1994; 269: 15388-15393Abstract Full Text PDF PubMed Google Scholar). Meprins are also expressed in leukocytes of the lamina propria of human inflammatory sites and in mouse mesenteric lymph nodes during intestinal inflammation, and they are found at high concentrations in human urine in women with urinary tract infections (5Bond J.S. Matters G.L. Banerjee S. Dusheck R.E. FEBS Lett. 2005; 579: 3317-3322Crossref PubMed Scopus (76) Google Scholar, 7Lottaz D. Hahn D. Muller S. Muller C. Sterchi E.E. Eur. J. Biochem. 1999; 259: 496-504Crossref PubMed Scopus (70) Google Scholar, 9Crisman J.M. Zhang B. Norman L.P. Bond J.S. J. Immunol. 2004; 172: 4510-4519Crossref PubMed Scopus (68) Google Scholar). The localization of meprins at the interface with the external environment, in leukocytes at inflammatory sites, and in response to bacterial infections implicates them in host defense. Furthermore, the observation that leukocytes from mice lacking the meprin β gene have a diminished ability to disseminate through the extracellular matrix indicates that meprins affect leukocyte infiltration (9Crisman J.M. Zhang B. Norman L.P. Bond J.S. J. Immunol. 2004; 172: 4510-4519Crossref PubMed Scopus (68) Google Scholar). In vitro studies have demonstrated that meprins are capable of cleaving extracellular matrix proteins such as laminin, collagens IV and V, fibrinogen, and cytokines such as osteopontin, bradykinin, and MCP-1 (monocyte chemotactic protein-1) (10Kruse M.N. Becker C. Lottaz D. Kohler D. Yiallouros I. Krell H.W. Sterchi E.E. Stocker W. Biochem. J. 2004; 378: 383-389Crossref PubMed Scopus (145) Google Scholar, 11Bertenshaw G.P. Turk B.E. Hubbard S.J. Matters G.L. Bylander J.E. Crisman J.M. Cantley L.C. Bond J.S. J. Biol. Chem. 2001; 276: 13248-13255Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 12Ambort D. Stalder D. Lottaz D. Huguenin M. Oneda B. Heller M. Sterchi E.E. FEBS J. 2008; 275: 4490-4509Crossref PubMed Scopus (10) Google Scholar). Thus, meprins are capable of cleaving a variety of substrates that are intimately involved in the host response to injury. Meprins are composed of two multidomain subunits (α and β) that are encoded on different chromosomes (α on human chromosome 6 and mouse chromosome 17; β on chromosome 18 in humans and mice) and share ∼40% amino acid identity. Despite the fact that the subunits are evolutionarily related, they have different substrate specificities and different structural properties. For example, the meprin β subunit has a preference for cleavage of peptide bonds flanked by negatively charged amino acids, whereas the meprin α subunit cleaves at bonds flanked by small or hydrophobic amino acids (11Bertenshaw G.P. Turk B.E. Hubbard S.J. Matters G.L. Bylander J.E. Crisman J.M. Cantley L.C. Bond J.S. J. Biol. Chem. 2001; 276: 13248-13255Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Both subunits contain signal sequences that direct the proteins to the secretory pathway, prosequences that keep the enzymes inactive, proteolytic domains of the “astacin family of metalloproteinases,” and other C-terminal non-catalytic domains (13Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (356) Google Scholar). The subunits form homo- or heteromeric dimers that are linked by disulfide bonds (14Ishmael F.T. Shier V.K. Ishmael S.S. Bond J.S. J. Biol. Chem. 2005; 280: 13895-13901Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). During biosynthesis, the meprin α subunit is proteolytically cleaved near the C terminus and thus loses its epidermal growth factor-like, transmembrane, and cytoplasmic domains. Thus, dimers of meprin α are secreted into the extracellular space. The meprin β subunit retains its epidermal growth factor, transmembrane, and cytoplasmic domains and moves to the plasma membrane as a type 1 membrane-bound protein (15Hengst J.A. Bond J.S. J. Biol. Chem. 2004; 279: 34856-34864Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). As a consequence, homo-oligomers of meprin α (called homomeric meprin A) are secreted into the extracellular space; heterodimers of meprins α and β (heteromeric meprin A) form tetramers and are membrane-bound; and homo-oligomers of meprin β form membrane-bound dimers (meprin B) (5Bond J.S. Matters G.L. Banerjee S. Dusheck R.E. FEBS Lett. 2005; 579: 3317-3322Crossref PubMed Scopus (76) Google Scholar, 16Bertenshaw G.P. Norcum M.T. Bond J.S. J. Biol. Chem. 2003; 278: 2522-2532Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The interactions of meprin isoforms with cytokines have not been studied systematically. It has been demonstrated, however, that meprin B is capable of activating IL-1β in vitro (17Herzog C. Kaushal G.P. Haun R.S. Cytokine. 2005; 31: 394-403Crossref PubMed Scopus (64) Google Scholar). Because of the structural similarity between IL-1β and IL-18 and the importance of these cytokines in inflammatory processes, we chose to determine whether the latter is also a substrate for meprin β in the heteromeric isoform of meprin A and homomeric meprin B. Furthermore, studies aimed to determine whether meprins generate biologically active forms of the cytokine using a cell-based functional assay that tested the ability of meprin β-processed IL-18 to activate NF-κB. In addition, we asked whether mice lacking the meprin β gene produce altered levels of active IL-18 in an experimental model of inflammatory bowel disease (IBD) induced by dextran sulfate sodium (DSS). Several groups have reported high levels of IL-18 in patients with active IBD, and this observation is mirrored in various mouse models of experimental IBD, where intestinal damage can be attenuated by blocking IL-18 (18Monteleone G. Trapasso F. Parrello T. Biancone L. Stella A. Iuliano R. Luzza F. Fusco A. Pallone F. J. Immunol. 1999; 163: 143-147PubMed Google Scholar, 19Wiercinska-Drapalo A. Flisiak R. Jaroszewicz J. Prokopowicz D. World J. Gastroenterol. 2005; 11: 605-608Crossref PubMed Scopus (32) Google Scholar, 20Sivakumar P.V. Westrich G.M. Kanaly S. Garka K. Born T.L. Derry J.M. Viney J.L. Gut. 2002; 50: 812-820Crossref PubMed Scopus (225) Google Scholar, 21Kanai T. Watanabe M. Okazawa A. Sato T. Yamazaki M. Okamoto S. Ishii H. Totsuka T. Iiyama R. Okamoto R. Ikeda M. Kurimoto M. Takeda K. Akira S. Hibi T. Gastroenterology. 2001; 121: 875-888Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). The results indicate that the meprin β subunit of heteromeric meprin A and homomeric meprin B is able to generate a physiologically active form of IL-18 and that this subunit affects the levels of active IL-18 in vivo in a mouse model of IBD. Expression and Purification of ProIL-18—Full-length mouse IL-18 containing an N-terminal histidine tag with a thrombin cleavage site in the linker region was constructed in a bacterial expression system. ProIL-18 obtained as pCR3.1::IL-18 (a gift from Camille Locht, Institute Pasteur de Lille) was cloned into pET30::IL-18 using NcoI-EcoRI restriction enzyme sites. The induced protein showed anomalous mobility and, upon further purification, gave unsatisfactory yield. Therefore, proIL-18 was subcloned into pET28a. NcoI of pET30::IL-18 was mutated to an NdeI site by site-directed mutagenesis using primers 5′-CGA CGA CGA CGA CAA CAT ATG GCT GCC ATG TCA G-3′ (sense) and 5′-C TGA CAT GGC AGC CAT ATG TTG TCG TCG TCG to generate ProIL-18 was and into using to generate proIL-18 with an N-terminal histidine tag was into the and from bacterial with 1 using acid matrix containing were and with using a to the N-terminal linker as as the histidine was by the protein to of Protein were to and by or The protein concentrations were using a of forms of mouse meprin α meprin meprin β (meprin B) and meprin (heteromeric meprin A) were from transfected cells the proteins as G.P. J.A. Bond J.S. Biol. Chem. 2002; PubMed Scopus Google Scholar, G.P. Bond J.S. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). α and β subunits are secreted as of homomeric meprin A and meprin B was an and 1 of heteromeric meprin A was a isoforms were activated by by at for 1 and through a to of meprin α and β subunits were using their peptide (a and and as was used to the of both subunits G.P. J.A. Bond J.S. Biol. Chem. 2002; PubMed Scopus Google Scholar). of ProIL-18 by was with homomeric meprin A and heteromeric meprin A and meprin B μm or in and in a of for to at The was by of and the were in containing The were to and proteins were by or The were with IL-18 by and the signal was by using substrate The for the was For meprin B were in the or absence of at of the ProIL-18 of with meprin B for was to and proteins were by The cleavage product was and for mass the was with and in with mass The were with and acid and in The was added to a and to tandem mass The obtained were the by proIL-18 using and the was for using the with for The identified was further to by for of product of proIL-18 by cleavage by meprin isoforms was using ProIL-18 was with μm meprin B or heteromeric meprin A at in of in and for of the were to and product was using The of product were substrate using to Km and The and were further β and IL-18 in Madin-Darby cells were in with with and meprin β was transfected into cells to in a using β was activated by cells were with of in was added to 1 of and at for The was cells were with and of in was added in 1 of and for at cells were of proIL-18 in 1 of and at for were to the The culture were to to and with The cells were with and and proteins were on and with β NF-κB in EL-4 by was tested as a of NF-κB activation in EL-4 cells. EL-4 cells were in with The cells were with and in to a of 1 ProIL-18 and meprin B was for at and of meprin proIL-18 was added to 1 of EL-4 cells and for meprin B or proIL-18 at of were and protein was using a protein was used for the NF-κB activation assay using a and was at of cells as the for the NF-κB of IBD and of IL-18 by was induced in meprin α knock-out mice a meprin βKO mice a and their wild-type mice by administering in their for and on the (9Crisman J.M. Zhang B. Norman L.P. Bond J.S. J. Immunol. 2004; 172: 4510-4519Crossref PubMed Scopus (68) Google Scholar). groups the study from of the different groups were by of by and was by into an The was at for at to The serum was tested for active IL-18 levels using a IL-18 assay The in this assay the active form of The of this assay is was used for were of Meprins with was with of activated and the were by A and Both heteromeric meprin A and meprin B cleaved proIL-18 and a 17-kDa A and and or in the of proIL-18 or the IL-18 was when proIL-18 was with homomeric meprin A A and and in not In addition, showed meprin B to be capable of of the 17-kDa cleaved product heteromeric meprin A the A and and meprin B concentrations of μm or and during meprin B was capable of the 17-kDa product at lower meprin B concentrations the 17-kDa product a an of meprins A and the cleavage of IL-18 of ProIL-18 by of IL-18 product of the 17-kDa was by and The Km and and the for the were by the of product substrate using the by B had a lower Km and a for of proIL-18 compared with heteromeric meprin indicating and of proIL-18 B (meprin meprin A (meprin in a of the of of ProIL-18 by the site of proIL-18 the of proIL-18 cleaved by meprin B were by as A to the C terminus of IL-18 was found in the indicating that the cleavage an N-terminal from the of the the for of proIL-18 a which from a a cleavage on the C terminus and a cleavage on the The amino acid of the was identified by further it to by the of thus the was to be The identified cleavage is with substrate for meprin an in the (11Bertenshaw G.P. Turk B.E. Hubbard S.J. Matters G.L. Bylander J.E. Crisman J.M. Cantley L.C. Bond J.S. J. Biol. Chem. 2001; 276: 13248-13255Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The meprin B cleavage site was from and cleavage sites, as in Caspase-1 and PR-3, which to the meprin β cleavage activate proIL-18, whereas which cleaves to the meprin β cleavage is to IL-18 (1Fantuzzi G. Dinarello C.A. J. Clin. Immunol. 1999; 19: 1-11Crossref PubMed Scopus (423) Google Scholar). Therefore, it was important to determine meprin B affects IL-18 B and ProIL-18 in a determine whether proIL-18 be cleaved in a cell-based meprin β was transfected into cells. activation of meprin B as proIL-18 was added to the cell culture medium. The cell culture was and the of IL-18 was by IL-18 was in the cell culture from cells 1 and IL-18 was in the from cells, of and The from cells meprin B also levels of proIL-18 cells active meprin B IL-18 in the culture The meprin 17-kDa was not that it was by the cells further by meprin B or other B in of determine whether the meprin B cleavage product of proIL-18 was biologically EL-4 cells containing for IL-18 were EL-4 cells were with proIL-18 meprin B or proIL-18 with meprin B for and the cell were for NF-κB As IL-18 is capable of NF-κB H. M. T. Ikeda M. H. Kurimoto M. Biochem. PubMed Scopus Google NF-κB activation in the levels of active IL-18 EL-4 cells with proIL-18 showed a in NF-κB EL-4 cells. was a in NF-κB in EL-4 cells when proIL-18 with meprin B was added to the cells the two meprin IL-18 had NF-κB These results indicate that meprin B activated βKO of determine whether meprins affect the levels of IL-18 in experimental model of IBD was induced by administering to mice of different meprin and the serum levels of the pro-inflammatory cytokine were as both the and meprin βKO mice showed elevated levels of active IL-18 upon the was in the βKO mice, and the between the two was This was with the in vitro that showed that meprin B cleavage was capable of active In similar with the meprin αKO mice, the in the levels of active IL-18 was that in the mice This between the two groups is a that the meprin activation of proIL-18 is physiologically The identifies proIL-18 as a substrate for subunit of meprin B and heteromeric meprin A and that the results in a biologically active form of Furthermore, of the meprin β gene results in a in the serum levels of active IL-18 in response to intestinal indicating the functional significance of this in vivo. IL-18 is a for the of IBD in and the cytokine to the of that the of the inflammation. Thus, meprins be with caspase-1 and as important in the of active forms of this ProIL-18 is one of the meprin B substrates identified to (11Bertenshaw G.P. Turk B.E. Hubbard S.J. Matters G.L. Bylander J.E. Crisman J.M. Cantley L.C. Bond J.S. J. Biol. Chem. 2001; 276: 13248-13255Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, G.P. Bond J.S. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). a of the was found to be the meprin B with a Km of μm and a of G.P. J.A. Bond J.S. Biol. Chem. 2002; PubMed Scopus Google Scholar). The for proIL-18 found μm and are to for and of the peptide substrates found for other For example, the of for a substrate to its physiological is T. J. A. K. W. M. I. G.M. 2002; PubMed Scopus Google Scholar). substrate for has a of H. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). In addition, the of meprin B for proIL-18 are similar to of For meprin the Km for proIL-18 is 1.3 for the Km is The of caspase-1 is that of meprin compared with a K. H. G. K. K. H. K. Kurimoto M. T. Sato V. 275: PubMed Scopus Google Scholar). of the of the meprin β subunit showed in the active site that can form with in the substrates (11Bertenshaw G.P. Turk B.E. Hubbard S.J. Matters G.L. Bylander J.E. Crisman J.M. Cantley L.C. Bond J.S. J. Biol. Chem. 2001; 276: 13248-13255Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). In with its preference for in the meprin B cleaves proIL-18 between and activating the It is for a to peptide bonds at negatively charged however, are to this Thus, meprin B the ability to peptide bonds at with metalloproteinases such as and H. M. 2003; PubMed Google Scholar, L. C. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). have for a acid peptide and as these proteases can activate or Thus, peptide bonds in the N-terminal region of proIL-18 to be to but the of the proteases whether activation or results from Meprins, proteases of intestinal epithelial cells and mesenteric are likely for activating a of the proIL-18 secreted at intestinal sites as a of damage in IBD J.M. Bond J.S. Biochem. PubMed Scopus Google Scholar, J.A. J. W. Sterchi E.E. FEBS Lett. PubMed Scopus Google Scholar). the of the serum of IL-18 in and βKO mice in the experimental model of IBD, we that the β subunit of meprin isoforms to of the active is that proIL-18 is released from and cells and from cells, the to extracellular PR-3, a membrane-bound of the of has been to activate proIL-18 at the plasma membrane a S. A. T. T. H. A. K. K. H. H. J. Immunol. 2001; PubMed Scopus Google Scholar). in meprin βKO mice, is a in the serum levels of active IL-18 Meprins be for this or be involved in the of cells, to the In meprin αKO mice, meprin β is and meprin B be the isoform of meprins that are similar levels of expression of meprin β in and meprin αKO mice, Banerjee and J. S. and when both meprin α and β subunits are of the β subunit is found as heteromeric meprin A G.P. Norcum M.T. Bond J.S. J. Biol. Chem. 2003; 278: 2522-2532Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The observation that the meprin αKO mice have levels of serum IL-18 compared with the mice that meprin B is heteromeric meprin A in active The in vitro studies indicate that meprin B has a for proIL-18 compared with heteromeric meprin A and this the is that matrix metalloproteinases are intimately involved in cytokines in vivo in the Immunol. 2004; 4: PubMed Scopus Google Scholar). This that meprins can the of IL-18, a key cytokine in the inflammatory and into the that effects activation of Sterchi of and for for the cell-based S. of for of the and the of for with mass and of for