Ribosomal Protein S19 Interacts with Macrophage Migration Inhibitory Factor and Attenuates Its Pro-inflammatory Function

Abstract

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in the pathogenesis of inflammatory disorders such as infection, sepsis, and autoimmune disease. MIF exists preformed in cytoplasmic pools and exhibits an intrinsic tautomerase and oxidoreductase activity. MIF levels are elevated in the serum of animals and patients with infection or different inflammatory disorders. To elucidate how MIF actions are controlled, we searched for endogenous MIF-interacting proteins with the potential to interfere with key MIF functions. Using in vivo biotin-tagging and endogenous co-immunoprecipitation, the ribosomal protein S19 (RPS19) was identified as a novel MIF binding partner. Surface plasmon resonance and pulldown experiments with wild type and mutant MIF revealed a direct physical interaction of the two proteins (KD = 1.3 × 10-6 m). As RPS19 is released in inflammatory lesions by apoptotic cells, we explored whether it affects MIF function and inhibits its binding to receptors CD74 and CXCR2. Low doses of RPS19 were found to strongly inhibit MIF-CD74 interaction. Furthermore, RPS19 significantly compromised CXCR2-dependent MIF-triggered adhesion of monocytes to endothelial cells under flow conditions. We, therefore, propose that RPS19 acts as an extracellular negative regulator of MIF. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in the pathogenesis of inflammatory disorders such as infection, sepsis, and autoimmune disease. MIF exists preformed in cytoplasmic pools and exhibits an intrinsic tautomerase and oxidoreductase activity. MIF levels are elevated in the serum of animals and patients with infection or different inflammatory disorders. To elucidate how MIF actions are controlled, we searched for endogenous MIF-interacting proteins with the potential to interfere with key MIF functions. Using in vivo biotin-tagging and endogenous co-immunoprecipitation, the ribosomal protein S19 (RPS19) was identified as a novel MIF binding partner. Surface plasmon resonance and pulldown experiments with wild type and mutant MIF revealed a direct physical interaction of the two proteins (KD = 1.3 × 10-6 m). As RPS19 is released in inflammatory lesions by apoptotic cells, we explored whether it affects MIF function and inhibits its binding to receptors CD74 and CXCR2. Low doses of RPS19 were found to strongly inhibit MIF-CD74 interaction. Furthermore, RPS19 significantly compromised CXCR2-dependent MIF-triggered adhesion of monocytes to endothelial cells under flow conditions. We, therefore, propose that RPS19 acts as an extracellular negative regulator of MIF. A large body of evidence now shows that macrophage migration inhibitory factor (MIF) 2The abbreviations used are: MIF, migration inhibitory factor; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography; RPS19, ribosomal protein S19; MALDI, matrix-assisted laser desorption/ionization; TOF, time of flight; wt, wild type; GST, glutathione S-transferase; HAoEC, human aortic endothelial cells; Ni-NTA, nickel-nitrilotriacetic acid; co-IP, co-immunoprecipitation. activates a range of intracellular pathways and plays a key role in host immune and inflammatory responses (1Calandra T. Roger T. Nat. Rev. Immunol. 2003; 3: 791-800Crossref PubMed Scopus (1322) Google Scholar, 2Hoi A.Y. Iskander M.N. Morand E.F. Inflamm. Allergy Drug Targets. 2007; 6: 183-190Crossref PubMed Scopus (79) Google Scholar). Certain of the MIF inflammatory functions also have been proposed to be the result of the unusual enzymatic properties of the protein, namely tautomerase and oxidoreductase activities (3Swope M. Sun H.W. Blake P.R. Lolis E. EMBO J. 1998; 17: 3534-3541Crossref PubMed Scopus (182) Google Scholar, 4Al-Abed Y. Dabideen D. Aljabari B. Valster A. Messmer D. Ochani M. Tanovic M. Ochani K. Bacher M. Nicoletti F. Metz C.N. Pavlov V.A. Miller E.J. Tracey K.J. J. Biol. Chem. 2005; 280: 36541-36544Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 5Rosengren E. Bucala R. Aman P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar, 6Thiele M. Bernhagen J. Antioxid. Redox Signal. 2005; 7: 1234-1248Crossref PubMed Scopus (92) Google Scholar). Inhibition or deletion of MIF attenuates disease progression in experimental models such as atherosclerosis, arthritis, glomerulonephritis, sepsis, autoimmune encephalitis, and autoimmune diabetes (7Lan H.Y. Bacher M. Yang N. Mu W. Nikolic-Paterson D.J. Metz C. Meinhardt A. Bucala R. Atkins R.C. J. Exp. Med. 1997; 185: 1455-1465Crossref PubMed Scopus (257) Google Scholar, 8Bernhagen J. Calandra T. Mitchell R.A. Martin S.B. Tracey K.J. Voelter W. Manogue K.R. Cerami A. Bucala R. Nature. 1993; 365: 756-759Crossref PubMed Scopus (931) Google Scholar, 9Pan J.H. Sukhova G.K. Yang J.T. Wang B. Xie T. Fu H. Zhang Y. Satoskar A.R. David J.R. Metz C.N. Bucala R. Fang K. Simon D.I. Chapman H.A. Libby P. Shi G.P. Circulation. 2004; 109: 3149-3153Crossref PubMed Scopus (147) Google Scholar, 10Leech M. Metz C. Santos L. Peng T. Holdsworth S.R. Bucala R. Morand E.F. Arthritis Rheum. 1998; 41: 910-917Crossref PubMed Scopus (149) Google Scholar, 11Denkinger C.M. Denkinger M. Kort J.J. Metz C. Forsthuber T.G. J. Immunol. 2003; 170: 1274-1282Crossref PubMed Scopus (102) Google Scholar, 12Stosic-Grujicic S. Stojanovic I. Maksimovic-Ivanic D. Momcilovic M. Popadic D. Harhaji L. Miljkovic D. Metz C. Mangano K. Papaccio G. Al-Abed Y. Nicoletti F. J. Cell. Physiol. 2008; 215: 665-675Crossref PubMed Scopus (67) Google Scholar, 13Hoi A.Y. Hickey M.J. Hall P. Yamana J. O'Sullivan K.M. Santos L.L. James W.G. Kitching A.R. Morand E.F. J. Immunol. 2006; 177: 5687-5696Crossref PubMed Scopus (117) Google Scholar). A pivotal step in the inflammatory response is the chemokine-governed adherence of monocytes to the endothelial lining which is then followed by their egress from the vasculature at the affected site. Earlier data from MIF-/- mice illustrate a role of MIF in leukocyte recruitment that was recently substantiated by the finding that MIF serves as a chemoattractant for monocytes and T cells by directly binding to the chemokine receptors CXCR2 and CXCR4 (14Bernhagen J. Krohn R. Lue H. Gregory J.L. Zernecke A. Koenen R.R. Dewor M. Georgiev I. Schober A. Leng L. Kooistra T. Fingerle-Rowson G. Ghezzi P. Kleemann R. McColl S.R. Bucala R. Hickey M.J. Weber C. Nat. Med. 2007; 13: 587-596Crossref PubMed Scopus (927) Google Scholar, 15Gregory J.L. Morand E.F. McKeown S.J. Ralph J.A. Hall P. Yang Y.H. McColl S.R. Hickey M.J. J. Immunol. 2006; 177: 8072-8079Crossref PubMed Scopus (144) Google Scholar). On the cell surface MIF also associates with CD74 (invariant chain of major histocompatibility complex class II) which colocalizes with CXCR2 (14Bernhagen J. Krohn R. Lue H. Gregory J.L. Zernecke A. Koenen R.R. Dewor M. Georgiev I. Schober A. Leng L. Kooistra T. Fingerle-Rowson G. Ghezzi P. Kleemann R. McColl S.R. Bucala R. Hickey M.J. Weber C. Nat. Med. 2007; 13: 587-596Crossref PubMed Scopus (927) Google Scholar, 16Leng L. Metz C.N. Fang Y. Xu J. Donnelly S. Baugh J. Delohery T. Chen Y. Mitchell R.A. Bucala R. J. Exp. Med. 2003; 197: 1467-1476Crossref PubMed Scopus (815) Google Scholar). Interaction with different surface molecules is thought to partly explain the wide impact of MIF on cellular pathways. Despite its role as a key mediator in immune and inflammatory diseases, very little is known of how MIF action is regulated and terminated. Accordingly, we searched for endogenous molecules with the ability to control key steps of MIF signaling (i.e. receptor binding and/or receptor-associated functions). In this study, we identified ribosomal protein S19 (RPS19), a component of the small ribosomal subunit that is also released by apoptotic cells (17Nishimura T. Horino K. Nishiura H. Shibuya Y. Hiraoka T. Tanase S. Yamamoto T. J. Biochem. (Tokyo). 2001; 129: 445-454Crossref PubMed Scopus (54) Google Scholar), as such a candidate. Identification of MIF-interacting Partners by Coimmunoprecipitation-Mouse NIH 3T3 fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum (PAA Laboratories, Cölbe, Germany) and antibiotics. Cells were lysed in Nonidet P-40 (1% IGEPAL CA-630) buffer containing protease inhibitors. Lysed cells were disrupted by passage through a 21-gauge needle and subjected to sonication by two 10-s bursts at 200-300 watts separated by a 10-s cooling period. After centrifugation at 12,000 × g at 4 °C for 10 min, the supernatant was precleared with 30 μl of protein G-Sepharose 4B Fast Flow on a at 4 °C for with MIF or serum on 30 μl of protein G-Sepharose at 4 °C for After with buffer for 10 were in buffer and for 10 were separated on a and with Identification of MIF-interacting Partners by in of of E. P. E. J. E. A. F. J. S. A. 2003; PubMed Scopus Google was the modified by G. as for in Scholar). MIF was by the in and the in and a M. and of Macrophage as was by the E. P. E. J. E. A. F. J. S. A. 2003; PubMed Scopus Google and were with and and or NIH 3T3 cells with and and was in steps to and 4 After were small in were for of a MIF protein with and of the that strongly the MIF protein and control were in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf and at Cells were lysed in IGEPAL and and to μl of of cell After on a were with proteins were by for 10 in separated by on a and with were and proteins in were with A. M. M. Chem. 1996; PubMed Scopus Google Scholar). were separated and by liquid on a of under conditions. were identified by in the data as Identification of RPS19 by and identified by was from the was with and with and were in a of a 10 in and for at were with μl of containing μl of the were to a of on an After 10 the supernatant was and the was with μl of of were by an identified protein was by in and were also to and of and MIF MIF that from MIF by MIF, MIF, was and as K. M. Meinhardt A. J. Mol. Cell. Biochem. 2008; PubMed Scopus Google Scholar). was which was also to the wild type human MIF MIF and MIF mutant proteins were from of of with for wild type MIF and MIF for MIF and MIF by R. Rorsman H. E. R. Bernhagen J. J. Biochem. PubMed Scopus Google Scholar). was with for Cells were and a was as J. Scholar). were on by 10-s bursts a Germany) and at × g at 4 °C for 30 and MIF and MIF were from the of the the MIF mutant was an body MIF mutant was in in and body proteins were with in PBS, and on a an were and directly In MIF and MIF proteins are in the with the of binding to the the MIF mutant the of the was in of 10 and 4 in the were in in protein was in PBS, by and directly as proteins a of as by of proteins was by as MIF, and MIF the to a as F. S. 3: PubMed Scopus Google Scholar), the MIF mutant this of the the mutant be and of and RPS19 was from RPS19 was by with and for the glutathione and and for the are and were of an the RPS19 was with with and to the were by and were in E. by with at °C for the RPS19 protein, cells were lysed in by sonication and with for 30 After centrifugation at 12,000 × g for min, the supernatant containing was subjected to 4B of the protein was as by the protein, cells were lysed with followed by and the protein was from the by protein was with and PBS, containing and Surface were on a MIF in was to a by the to the levels of were response response to 10 on a were at °C and as a buffer at a flow of experiments were at °C at a flow of μl of the (RPS19) in buffer were the MIF followed by a buffer was the of RPS19 were the and of RPS19 and MIF and RPS19 or wild type MIF were in In the of serum were RPS19 on J. M.J. and Google Scholar). RPS19 were for and at 4 A was from L. G. P. A. J. C. J.A. J. N. 2003; PubMed Google Scholar). MIF is through Cells were on and with for 10 for in serum and serum was followed by with RPS19 with with and by NIH by with were at 4 and were for at was used for were with a laser of MIF was the protein to the of the was separated from MIF MIF was with PBS, and were on 30 μl of by in μl of at on a for 30 To MIF, were with and then in μl of buffer IGEPAL CA-630) with of and on a at 4 °C for As a were with the of were then with buffer and in buffer for were separated by and with with was on with PBS, and for with different of MIF or of human wild type MIF or MIF, MIF, and MIF As a MIF proteins were with After with buffer as or buffer IGEPAL in the of human MIF and MIF proteins to the were in by and with or and for RPS19, and for MIF. was as K. Al-Abed Y. D.J. Peng T. Calandra T. Metz C.N. Bucala R. 1997; PubMed Scopus Google Scholar). was in an by μl of containing at a of with μl of In that MIF and RPS19, proteins were in μl of for As F. A. M. A. J. Mol. 2005; PubMed Scopus Google Scholar), a protein of and with the was was by the at in an in and to a of the a time of the in from to 4 the was and as tautomerase activity. were to the with a of were Flow aortic endothelial cells Germany) were in medium and used at cells of H. W. L. of were in medium supplemented with 10% fetal calf and 10 human as C. M. H. H.W. Weber J. Immunol. 1993; PubMed Scopus Google Scholar). flow were as (14Bernhagen J. Krohn R. Lue H. Gregory J.L. Zernecke A. Koenen R.R. Dewor M. Georgiev I. Schober A. Leng L. Kooistra T. Fingerle-Rowson G. Ghezzi P. Kleemann R. McColl S.R. Bucala R. Hickey M.J. Weber C. Nat. Med. 2007; 13: 587-596Crossref PubMed Scopus (927) Google Scholar). were grown to in and with MIF and or control buffer for at were at the of a flow and on the of an with and cells × with were with a CXCR2 or control and in buffer × 10 serum cell was supplemented at °C with × the flow at a of for of monocytes was in the cell are as the were to experimental with a of were was as D. S. Leng L. Y. Y. J. C. M. Mitchell R.A. J. Chen Y. H. J. M. D. J. Bernhagen J. Lolis E. Bucala R. J. Immunol. 2008; PubMed Scopus Google Scholar). were with of CD74 at 4 °C After 4 with the were with at 4 °C of RPS19 were with from for at in the was and with of the RPS19 and was in the at 4 °C for After the 4 of was for at in the followed by of the of and to in the at and at human MIF was by at °C for Identification of RPS19 as a MIF-interacting was under to for the of MIF-interacting As cell type NIH or and a of experiments were with endogenous of NIH 3T3 In with a MIF a of MIF-interacting proteins were A at was identified in the in a with an control protein was by followed by and identified as ribosomal protein S19 of this finding was the were and with an RPS19 and then with an MIF proteins were in the the was was RPS19 also be identified as a MIF-interacting protein in a different a MIF protein that was in NIH 3T3 was a that is and in vivo by the that was in the NIH 3T3 E. P. E. J. E. A. F. J. S. A. 2003; PubMed Scopus Google Scholar). MIF and MIF-interacting proteins were in a step by binding to and separated by A NIH 3T3 that was used as were and proteins were identified by RPS19 was identified the proteins that were from cells MIF from cells the finding that two RPS19 as an MIF-interacting protein evidence for a interaction of MIF with RPS19 in were to whether the interaction MIF and RPS19 was direct or on was with of As a were with the of of by that MIF directly with RPS19, and the RPS19 was the was to and from the of this interaction was by a MIF and RPS19 and a of MIF or were the were to with MIF. RPS19 was with MIF, RPS19 be the the interaction of RPS19 with MIF In an pulldown RPS19 was on and with of MIF or of human MIF. to the were separated by and with MIF to the was on RPS19 also directly with human MIF which is to the To the of the we also used MIF in the pulldown MIF, MIF, and R. Rorsman H. E. R. Bernhagen J. J. Biochem. PubMed Scopus Google Scholar). the MIF mutant was the wild type MIF, the MIF and MIF were to of the of the complex were surface plasmon data were used to the = × × and = × and the = 1.3 × 10-6 4 × In interaction of MIF with RPS19 has been by namely endogenous co-IP, in vivo pulldown and surface plasmon MIF and RPS19 in NIH 3T3 of NIH 3T3 cells that MIF with RPS19 of is in the with in the (RPS19) or (MIF) was in the MIF and RPS19 cellular a in MIF by the tautomerase of MIF a role in of its functions as a cytokine Lolis E. Rev. Physiol. Biochem. PubMed Google Scholar), we RPS19 binding to MIF its enzymatic activity. of to by MIF was in the or of RPS19 of RPS19 in a of MIF tautomerase which was at a of RPS19 MIF at an of J. M. R. J. M. J. N. Y. PubMed Scopus Google Scholar), a protein of and enzymatic was used as MIF tautomerase was which was also at a MIF at a we that proteins have a on MIF tautomerase activity. RPS19 MIF to RPS19 interfere with the binding of MIF to its receptor a was of RPS19 to the interaction of with CD74 by at the the of RPS19 to which at doses As a of human MIF the binding of to CD74 by human MIF inhibitory the inhibitory of RPS19 at the was the of human MIF through CXCR2 by it was that MIF inflammatory and leukocyte recruitment through activities of chemokine receptors CXCR2 and CXCR4 (14Bernhagen J. Krohn R. Lue H. Gregory J.L. Zernecke A. Koenen R.R. Dewor M. Georgiev I. Schober A. Leng L. Kooistra T. Fingerle-Rowson G. Ghezzi P. Kleemann R. McColl S.R. Bucala R. Hickey M.J. Weber C. Nat. Med. 2007; 13: 587-596Crossref PubMed Scopus (927) Google Scholar). used this to the of cells be modified by cells were with a were with wild type human MIF in the of the of cells was from of the by control in a in the of cells After of with MIF and RPS19, the of cells by from was and at a of RPS19 MIF of to MIF and RPS19 the of cells to that of of cells by MIF as as MIF by RPS19 is In the has been in of the role MIF plays in cellular and in a of from infection to and R. Lolis E. Drug 2005; PubMed Scopus Google Scholar, E.F. M. Bernhagen J. Nat. Rev. Drug 2006; PubMed Scopus Google Scholar). In of MIF whether by or small has as a potential of M. Metz C. Bucala R. Morand E.F. Arthritis Rheum. PubMed Scopus Google Scholar, L. Hall P. Metz C. Bucala R. Morand E.F. Exp. Immunol. 2001; PubMed Scopus Google Scholar). As MIF functions the cytokine to control the and progression of an inflammatory MIF the of J.A. Bucala R. Med. Scopus Google Scholar). the of endogenous proteins that inhibit MIF is of Using an in vivo E. P. E. J. E. A. F. J. S. A. 2003; PubMed Scopus Google Scholar), the RPS19 was identified as a MIF-interacting in NIH 3T3 endogenous as an for the of we RPS19 as an MIF binding partner. of was by the of a MIF-interacting R. A. G. R. A. Roger T. Calandra T. A. M. D. H. Bernhagen J. Nature. PubMed Scopus Google physical interaction was by pulldown experiments of proteins and binding by surface plasmon of of the complex (KD = 1.3 × is the range of and for in intracellular or in the binding of to the T cell receptor or major histocompatibility that of the complex with a in the range is strongly of a also of the of MIF an of MIF Certain activities of MIF are known to be by its enzymatic (3Swope M. Sun H.W. Blake P.R. Lolis E. EMBO J. 1998; 17: 3534-3541Crossref PubMed Scopus (182) Google Scholar, R. Rorsman H. E. R. Bernhagen J. J. Biochem. PubMed Scopus Google Scholar, K. Al-Abed Y. D.J. Peng T. Calandra T. Metz C.N. Bucala R. 1997; PubMed Scopus Google Scholar). to whether MIF that activities impact on RPS19 MIF tautomerase is on its oxidoreductase is on the R. Rorsman H. E. R. Bernhagen J. J. Biochem. PubMed Scopus Google Scholar). that the MIF mutant a binding as the wild type protein, the and to that is for a direct interaction. the and are have their tautomerase R. Rorsman H. E. R. Bernhagen J. J. Biochem. PubMed Scopus Google Scholar), in binding to the of the RPS19 an of for that this is by the by the that is for which is by that RPS19 with MIF tautomerase activity. is in to the MIF which the of the cytokine Y. Dabideen D. Aljabari B. Valster A. Messmer D. Ochani M. Tanovic M. Ochani K. Bacher M. Nicoletti F. Metz C.N. Pavlov V.A. Miller E.J. Tracey K.J. J. Biol. Chem. 2005; 280: 36541-36544Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). as a component of the small subunit of the an of the protein RPS19 also exists in a in the F. T. S. F. G. Biochem. 2001; PubMed Scopus Google Scholar). As RPS19 has functions by its ability to interfere with factor signaling its with factor F. T. S. F. G. Biochem. 2001; PubMed Scopus Google Scholar, Biochem. 1996; Full Text PDF PubMed Scopus Google and the A. L. A. A. P. E. F. M. A. N. C. I. 2005; Google Scholar). A of proteins that were on identified a of proteins with and S. A. M. M. F. M. C. I. Mol. Cell. 2007; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). which identified RPS19 factor or MIF, be to the different to the (i.e. co-IP, in vivo a RPS19 has been as a factor in human released by apoptotic cells the extracellular H. Shibuya Y. S. Tanase S. T. Yamamoto T. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). MIF the RPS19 a on monocytes by the factor and binding as a to the receptor H. Shibuya Y. Yamamoto T. 1998; Google Scholar). to the of RPS19 in the of function which also and MIF (14Bernhagen J. Krohn R. Lue H. Gregory J.L. Zernecke A. 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A. R. G. J. S. 2006; PubMed Scopus Google Scholar). is to that by MIF cytokine RPS19 an inflammatory is by the finding that of RPS19 in a of MIF binding to its receptor CD74 with the of MIF to the of for by the of RPS19 on the recently function of MIF as a of chemokine receptors CXCR2 and CXCR4 (14Bernhagen J. Krohn R. Lue H. Gregory J.L. Zernecke A. Koenen R.R. Dewor M. Georgiev I. Schober A. Leng L. Kooistra T. Fingerle-Rowson G. Ghezzi P. Kleemann R. McColl S.R. Bucala R. Hickey M.J. Weber C. Nat. Med. 2007; 13: 587-596Crossref PubMed Scopus (927) Google Scholar). MIF was found to the recruitment of monocytes and T by with CXCR2 and a that also In flow RPS19 significantly the adhesion of monocytes to aortic endothelial cells, that RPS19 extracellular of MIF for receptor In which inhibits of was recently identified as an intracellular of MIF function H. H.A. H. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). In data that RPS19 functions as an extracellular of MIF. A in from MIF to a complex MIF function at of the of and autoimmune for the for for for for a of for at the for protein by and and Lue for