Human Macrophage Migration Inhibitory Factor

Abstract

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory mediator with the ability to induce various immunomodulatory responses and override glucocorticoid-driven immunosuppression. Some of these functions have been linked to the unusual enzymatic properties of the protein, namely tautomerase and oxidoreductase activities. However, there are conflicting reports regarding the functional role of these enzymatic properties in normal physiological homeostasis and disease progression. Therefore, we have produced a highly pure, virtually endotoxin-free recombinant MIF preparation and fully characterized this using a variety of biochemical and biophysical approaches. The recombinant protein, with demonstrable enzymatic activity, was then used to systematically examine the biological activity of MIF. Surprisingly, treatment with MIF alone failed to induce cytokine expression, with the exception of IL-8. However, co-treatment of lipopolysaccharide (LPS) in conjunction with MIF produced synergistic secretion of tumor necrosis factor-α, interleukin (IL)-1, and IL-8 compared with LPS alone. The potentiating effect of MIF was seen at physiologically relevant concentrations. These data suggest that MIF has no conventional cytokine activity but, rather, acts to modulate and amplify the response to LPS. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory mediator with the ability to induce various immunomodulatory responses and override glucocorticoid-driven immunosuppression. Some of these functions have been linked to the unusual enzymatic properties of the protein, namely tautomerase and oxidoreductase activities. However, there are conflicting reports regarding the functional role of these enzymatic properties in normal physiological homeostasis and disease progression. Therefore, we have produced a highly pure, virtually endotoxin-free recombinant MIF preparation and fully characterized this using a variety of biochemical and biophysical approaches. The recombinant protein, with demonstrable enzymatic activity, was then used to systematically examine the biological activity of MIF. Surprisingly, treatment with MIF alone failed to induce cytokine expression, with the exception of IL-8. However, co-treatment of lipopolysaccharide (LPS) in conjunction with MIF produced synergistic secretion of tumor necrosis factor-α, interleukin (IL)-1, and IL-8 compared with LPS alone. The potentiating effect of MIF was seen at physiologically relevant concentrations. These data suggest that MIF has no conventional cytokine activity but, rather, acts to modulate and amplify the response to LPS. MIF 2The abbreviations used are: MIF, macrophage migration inhibitory factor; AP-1, activator protein 1; Jab-1, Jun activation domain-binding protein 1; NOESY, nuclear Overhauser effect spectroscopy; PAG, proliferation-associated gene; TNF, tumor necrosis factor; LPS, lipopolysaccharide; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Ab, antibody; IL, interleukin; EGF, epidermal growth factor. is a highly evolutionarily conserved 12.5-kDa protein that was assigned a unique combination of hormone-like, cytokine, and thioredoxin-like properties (1Lue H. Kleemann R. Calandra T. Roger T. Bernhagen J. Microb. Infect. 2002; 4: 449-460Crossref PubMed Scopus (304) Google Scholar). Significant interest in MIF as a pro-inflammatory mediator involved in human disease was based on the following important findings: (i) raised MIF concentrations in peripheral blood and specific tissue specimens in a broad range of diseases, including inflammatory conditions, various tumors, and metabolic disorders such as atherosclerosis, diabetes, and obesity (2Lan H.Y. Mu W. Yang N. Meinhardt A. Nikolic-Paterson D.J. Ng Y.Y. Bacher M. Atkins R.C. Bucala R. Am. J. Pathol. 1996; 149: 1119-1127PubMed Google Scholar, 3Leech M. Metz C. Hall P. Hutchinson P. Gianis K. Smith M. Weedon H. Holdsworth S.R. Bucala R. Morand E.F. Arthritis Rheum. 1999; 42: 1601-1608Crossref PubMed Scopus (293) Google Scholar, 4Makita H. Nishimura M. Miyamoto K. Nakano T. Tanino Y. Hirokawa J. Nishihira J. Kawakami Y. Am. J. Respir. Crit. Care Med. 1998; 158: 573-579Crossref PubMed Scopus (115) Google Scholar, 5Rossi A.G. Haslett C. Hirani N. Greening A.P. Rahman I. Metz C.N. Bucala R. Donnelly S.C. J. Clin. Investig. 1998; 101: 2869-2874Crossref PubMed Scopus (196) Google Scholar, 6Shimizu T. Abe R. Nakamura H. Ohkawara A. Suzuki M. Nishihira J. Biochem. Biophys. Res. Commun. 1999; 264: 751-758Crossref PubMed Scopus (190) Google Scholar, 7Schober A. Bernhagen J. Thiele M. Zeiffer U. Knarren S. Roller M. Bucala R. Weber C. Circulation. 2004; 109: 380-385Crossref PubMed Scopus (156) Google Scholar); (ii) genetic evidence of linkage with juvenile idiopathic arthritis (8Donn R.P. Alourfi Z. Zeggini E. Lamb R. Jury F. Lunt M. Meaza C. De Benedetti F. Thompson W. BPRG Study GroupArthritis Rheum. 2004; 50: 1604-1610Crossref PubMed Scopus (119) Google Scholar), rheumatoid arthritis (9Ichiyama H. Onodera S. Nishihira J. Ishibashi T. Nakayama T. Minami A. Yasuda K. Tohyama H. Cytokine. 2004; 26: 187-194Crossref PubMed Scopus (61) Google Scholar); (iii) neutralization of MIF by anti-MIF antibodies, shown to be therapeutically beneficial in a variety of animal models of inflammatory diseases, including sepsis, rheumatoid arthritis, pulmonary infections, and atherosclerosis (4Makita H. Nishimura M. Miyamoto K. Nakano T. Tanino Y. Hirokawa J. Nishihira J. Kawakami Y. Am. J. Respir. Crit. Care Med. 1998; 158: 573-579Crossref PubMed Scopus (115) Google Scholar, 7Schober A. Bernhagen J. Thiele M. Zeiffer U. Knarren S. Roller M. Bucala R. Weber C. Circulation. 2004; 109: 380-385Crossref PubMed Scopus (156) Google Scholar, 9Ichiyama H. Onodera S. Nishihira J. Ishibashi T. Nakayama T. Minami A. Yasuda K. Tohyama H. Cytokine. 2004; 26: 187-194Crossref PubMed Scopus (61) Google Scholar, 10Calandra T. Echtenacher B. Roy D.L. Pugin J. Metz C.N. Hultner L. Heumann D. Mannel D. Bucala R. Glauzer M.P. Nat. Med. 2000; 6: 164-170Crossref PubMed Scopus (694) Google Scholar). MIF has no homology with any other pro-inflammatory cytokines, and the mechanism(s) by which MIF exerts its biological effects remain unclear. Attempts to identify a cell surface MIF transmembrane receptor, which would explain some of the reported MIF regulatory effects in relation to extracellular signal-regulated protein kinase-1/2 (11Mitchell R.A. Metz C.N. Peng T. Bucala R. J. Biol. Chem. 1999; 274: 18100-18106Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar), synovial cell p38 kinase (12Santos L.L. Lacey D. Yang Y. Leech M. Morand E.F. J. Rheumatol. 2004; 31: 1038-1043PubMed Google Scholar), and p53 (13Mitchell R.A. Liao H. Chesney J. Fingerle-Rowson G. Baugh J. David J. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 345-350Crossref PubMed Scopus (521) Google Scholar, 14Fingerle-Rowson G. Petrenko O. Metz C.N. Forsthuber T.G. Mitchell R. Huss R. Moll U. Muller W. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9354-9359Crossref PubMed Scopus (245) Google Scholar), have been unsuccessful. CD74 (invariant polypeptide of MHC type II) was found to be a putative MIF receptor (15Leng 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 (820) Google Scholar), although there is no compelling evidence of any potential link between this antigen-processing molecule and intracellular signaling pathways. The absence of a validated signal transduction mechanism via a transmembrane receptor suggests that MIF may mediate its effects mainly by non-receptor mediated endocytosis (16Kleemann R. Grell M. Mischke R. Zimmermann G. Bernhagen J. J. Interferon Cytokine Res. 2002; 22: 351-363Crossref PubMed Scopus (34) Google Scholar). In contrast to all other known cytokines, MIF has several unusual intrinsic enzymatic activities, specifically l-dopachrome tautomerase, phenylpyruvate tautomerase (17Rosengren E. Bucala R. Aman P. Jacobsson L. Odh G. Metz C.N. Rorsmann H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar), and thiol-protein oxidoreductase (18Kleemann R. A. A. Mischke R. O. S. H. Bernhagen J. J. Mol. Biol. 1998; PubMed Scopus Google activities. MIF as a in physiologically relevant MIF tautomerase activity has to be using of MIF tautomerase activity have produced and have of tautomerase enzymatic activity with any biological functions K. Y. Peng T. Calandra T. Metz C.N. Bucala R. PubMed Scopus Google Scholar, A. S. 1999; PubMed Scopus Google Scholar, S. K. Y. Y. M. Nishihira J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, M. E. J. 1998; Scopus Google Scholar). of molecule with inhibitory properties the tautomerase activity of MIF have been ability to the properties of MIF M. S. N. S. 2002; PubMed Scopus Google Scholar). MIF activity via its that to R. H. E. Mischke R. Bernhagen J. J. Biochem. 2000; PubMed Scopus Google Scholar, R. A. G. Mischke R. A. O. Roger T. Calandra T. A. Grell M. D. H. Bernhagen J. 2000; PubMed Scopus Google and the protein proliferation-associated H. T. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of MIF the activity and activity in J. H. H. Kleemann R. P. A. Bernhagen J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). the between these and the reported cytokine of MIF of cytokine using recombinant MIF have produced conflicting MIF was shown to induce secretion by and J. H. H. Kleemann R. P. A. Bernhagen J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Bernhagen J. Metz C.N. Bacher M. Donnelly T. A. Bucala R. PubMed Scopus Google Scholar, Y. Metz C.N. F. Mitchell R.A. Chesney J. J. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar), and by K. E. Metz C.N. M. T. P. E. David C. Nat. 2: PubMed Scopus Google Scholar), and and IL-8 by synovial (12Santos L.L. Lacey D. Yang Y. Leech M. Morand E.F. J. Rheumatol. 2004; 31: 1038-1043PubMed Google Scholar, S. Nishihira J. Y. T. Y. H. Ishibashi T. Minami A. Arthritis Rheum. 2004; 50: PubMed Scopus Google Scholar). In K. E. Metz C.N. M. T. P. E. David C. Nat. 2: PubMed Scopus Google identify any effect of MIF on in a K. E. Metz C.N. M. T. P. E. David C. Nat. 2: PubMed Scopus Google Scholar). MIF human cell cytokine K. T. K. T. M. K. 1999; PubMed Scopus Google Scholar, T. H. N. M. A. Y. R. Nakano T. T. K. J. 1999; Google Scholar, H. R. A. N. M. A. K. Y. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). be that in these reports the protein was as inhibitory although this has to human MIF. The to the in effects found in is and endotoxin-free recombinant MIF. there have been in the of LPS reported in of MIF T. Bernhagen J. Metz C.N. Bacher M. Donnelly T. A. Bucala R. PubMed Scopus Google Scholar, J. Mitchell R.A. Calandra T. W. A. Bucala R. PubMed Scopus Google Scholar, A. J. B. B. Mitchell R. E. Y. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google and a of of LPS by some J. H. H. Kleemann R. P. A. Bernhagen J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, R. C. D. S. S. J. 2003; PubMed Scopus Google Scholar, D. A. Mitchell R. Bucala R. L. Leech M. Morand E. Arthritis Rheum. 2003; PubMed Scopus Google Scholar). these MIF has been as a cytokine in its and the of LPS in recombinant MIF as a of response in various in and in models has been to the reported MIF that have the of a virtually endotoxin-free protein preparation and of the The protein was characterized using enzymatic and biophysical approaches. we the biological to MIF and to conflicting reports regarding its MIF was by human using the following and and The was with and and the to The was by of the to the of The G. has been S. E. W. G. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The was The was by the human to to the of the in the The has been W. M. C. J. J. S. T. J. S. H. H. D. S. Mol. Biol. PubMed Scopus Google Scholar). and in with fetal in in with and induce to a macrophage in the of and with recombinant MIF LPS, at a of in and with and was with and in human in PBS, and at in in and by of at and in of recombinant macrophage factor at a of using and in in with FBS, and used synovial with rheumatoid arthritis as S. K. Y. Y. M. Nishihira J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google and used at The was to of The of the was as by The of the was as by at in the of with in with and of and of recombinant human MIF was as J. Mitchell R.A. Calandra T. W. A. Bucala R. PubMed Scopus Google with some with to of and with to a of at the in PBS, and in at of the of growth in of protein and and at was by at The was and a with of The recombinant MIF was using a with a of and to a with The MIF, was using of the MIF was as by with using endotoxin-free and cell was to LPS the MIF. was with of by of to all and with of MIF was and with the LPS of the MIF protein was using and using in The of LPS in this MIF was to LPS MIF was to a using on a The was with of then with and with and with MIF was the in and at The was a The MIF was by protein and by at The MIF was as by on preparation was using the as and and by using The of recombinant MIF was by using protein following the MIF was by using human MIF following of MIF the and using the and of at in cell by to the using the to the in of was cell and of was used in concentrations of signal was using was using and of was using a to the on by to with in and in in of MIF was using MIF at and by in a CD74 in was using CD74 at a of of MIF, namely and PAG, using a of and a of in was using a at a of was used in was used in on a at The of MIF was at MIF in protein and of MIF at in with on a at The of the was with in and to In the in the was and was used activity of MIF was as K. Y. Peng T. Calandra T. Metz C.N. Bucala R. PubMed Scopus Google Scholar). of l-dopachrome was by of with of and and The was the l-dopachrome by a the was with of l-dopachrome was with of was used in the enzymatic activity was in the of of was with of the l-dopachrome the was recombinant human MIF was of of at was The specific activity of human MIF was tautomerase bovine with to human MIF. of enzymatic activity of MIF was to the (18Kleemann R. A. A. Mischke R. O. S. H. Bernhagen J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar, A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the is based on the of and of the The of was at The was in a of The was by recombinant MIF to the of was compared the by recombinant human in the of of MIF, LPS, and on Cytokine PBMC, and synovial with concentrations of recombinant MIF and LPS at activity of MIF, PBMC, and with in the absence of MIF with LPS and effects of MIF and LPS using concentrations of LPS and of with various concentrations of MIF. of and cell using the using with and and to between and was found in this of with the and in the of the of activity by The cell that the activity was of growth factor was used as a of the response the of this growth factor is known to the Chen Z. J. S. C. C. J. 2002; 99: PubMed Scopus Google Scholar). in in activity, in with by with with recombinant MIF, a combination of the of of activity, cell with and of was and the signal was on a at in to The response was of with and using to the in and by and by and was in these the effect of MIF with the in using as the in and to treatment as that all the the and the used in in a in with and in in and as and was by activity and by of a activity was using the and of and cell was by of cell using and as D. A. P. G. Nat. Biol. 1999; PubMed Scopus Google Scholar). and with recombinant MIF specific was in in was by LPS a in using and the of found in was all of in and bovine at in the of to to by of with at using a with of at and at with was all data are as the there was some between in the of cytokine by was to data data The was using of have human recombinant MIF in E. to conflicting reports regarding the biological of this of the E. we have and LPS MIF that the to and a the of recombinant MIF was the was used to any and LPS to MIF. The preparation of of recombinant MIF of which the that biological effects be to LPS activity of MIF the of in this MIF preparation reports S. K. Y. Y. M. Nishihira J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Mitchell R.A. Calandra T. W. A. Bucala R. PubMed Scopus Google Scholar). of MIF was by protein was with anti-MIF by to the been and The of this protein was by The of recombinant MIF was by The data that was that to that found R. H. E. Mischke R. Bernhagen J. J. Biochem. 2000; PubMed Scopus Google Scholar), the of this MIF of MIF and in the was its ability to tautomerase and oxidoreductase The tautomerase activity of MIF was with l-dopachrome and a the specific activity was with the specific activity of bovine tautomerase (17Rosengren E. Bucala R. Aman P. Jacobsson L. Odh G. Metz C.N. Rorsmann H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar). MIF activity with a specific activity of recombinant MIF of using as The of MIF, to of that MIF as a with a H. M. A. I. Suzuki M. Nishihira J. 1999; PubMed Scopus Google Scholar, 1999; PubMed Scopus Google Scholar). The has and has by to the that MIF as a in was using Yang M. Biophys. Chem. 2004; PubMed Scopus Google Scholar). In of the protein using and a the of MIF and are with reported in (18Kleemann R. A. A. Mischke R. O. S. H. Bernhagen J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar, M. E. J. 1998; Scopus Google Scholar). The of MIF that MIF preparation the as other recombinant and of MIF T. Bernhagen J. Metz C.N. Bacher M. Donnelly T. A. Bucala R. PubMed Scopus Google Scholar). the and of MIF the the of a protein, the of this In with reports M. E. J. 1998; Scopus Google Scholar), the was a 12.5-kDa protein and with the of MIF Yang M. Biophys. Chem. 2004; PubMed Scopus Google has of human MIF. of MIF at in on a at The is the of a MIF in with on a at The in the was and was used MIF and Cytokine of synovial PBMC, and with to highly MIF in any in the of and data was no of secretion and with MIF. we any effect of MIF on synovial although the However, recombinant MIF a of IL-8 in with and The effect of MIF at a physiologically relevant MIF was of IL-8 by human no IL-8 response to MIF MIF the LPS any effect of MIF on the LPS response in synovial However, there was a effect of MIF on of and by human was in the of MIF and LPS The synergistic effects found in relation to in the of MIF with LPS of was to as a of of and by Y. Metz C.N. F. Mitchell R.A. Chesney J. J. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar, T. R. L. Bucala R. PubMed Scopus Google Scholar). and with recombinant MIF by with concentrations of LPS. of with this effect was in the of of MIF. concentrations of MIF no effect with MIF and of the inhibitory effect on and by and any effect of MIF on the of IL-8 in any cell type of with recombinant MIF in any of IL-8 in to with LPS there was no effect of MIF on the in and MIF effects on by AP-1, using a cell with activity of in MIF alone no effect on activity in these and the of the response was with of activity in this MIF the of the effect in with the reported activity of this factor In no effect of MIF was seen on of in and MIF activation of a In a range of concentrations was in and no activity of MIF has been seen in MIF on and have as in of and treatment with MIF. no in treatment with LPS, MIF, a combination of MIF and LPS. the of no effect on was of treatment with LPS, MIF and LPS, MIF, and which some of of MIF a effect on of in PBMC, as of MIF to to of in the of of with the range of concentrations of MIF in the of the of LPS effect on effect on was seen with MIF with a LPS MIF of with LPS MIF LPS and MIF concentrations to any effect on cell and any in surface CD74 in with recombinant MIF of MIF on and MIF was shown to of R. C. D. S. S. J. 2003; PubMed Scopus Google Scholar), the LPS in preparation of recombinant MIF. have the potential effect of MIF on cell and and peripheral blood with MIF and by cell the of was no in of in of In recombinant MIF alone in combination with IL-8 and migration of peripheral blood MIF has been as a cytokine, MIF has been as a cytokine although its in the of normal concentrations MIF has homology with several is a that is by MIF enzymatic The of the enzymatic of MIF with its biological role is is that some immunomodulatory effects of MIF in be by LPS of recombinant have in of MIF biological MIF these we have produced highly endotoxin-free recombinant MIF. MIF in this LPS some reports S. K. Y. Y. M. Nishihira J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Mitchell R.A. Calandra T. W. A. Bucala R. PubMed Scopus Google Scholar). preparation of MIF failed to induce the of any cytokine with the exception of IL-8. In contrast to S. K. Y. Y. M. Nishihira J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Nishihira J. Y. T. Y. H. Ishibashi T. Minami A. Arthritis Rheum. 2004; 50: PubMed Scopus Google Scholar), we IL-8 in synovial with MIF. The cell type in and to recombinant MIF by cytokine in MIF has a synergistic effect with concentrations of LPS on and of treatment with the MIF to override the of MIF the of and by LPS. The effect was at concentrations of MIF, to that inflammatory The to was seen using In these recombinant MIF, at was to of the The effects of MIF on specific to these there was no effect seen on of a and no of a The potential role of MIF as a to modulate the and of was effects seen on These that MIF and may the of the inflammatory this effect of MIF is with LPS and has no of its to any cytokine with exception of IL-8. The ability of MIF to to LPS is by the MIF ability to of of pro-inflammatory has been reported that MIF acts to of activity, important factor in of cytokine is known that MIF activity, receptor and R. A. G. Mischke R. A. O. Roger T. Calandra T. A. Grell M. D. H. Bernhagen J. 2000; PubMed Scopus Google Scholar). MIF was found to via its with the protein PAG, a protein, known as I. tautomerase and oxidoreductase of MIF and activity of H. T. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). unusual with surface of highly conserved of J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google with MIF H. T. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and to cell and In and via with J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). that of of in of effects on cell growth and O. Moll Mol. Full Text Full Text PDF PubMed Scopus Google Scholar). In we the effect of recombinant MIF the of in of MIF. suggest that regulatory link between MIF, PAG, and is and other is evidence these and data that recombinant MIF acts to of MIF may activity with the that we any in of with MIF, there are of between MIF and on response to and LPS. MIF has a effect on on of LPS In LPS MIF of of LPS to of in the of MIF. These data with activity of MIF on and in the of the of regulatory link between MIF and and that of may have functional on of responses to and LPS in relation to cytokine and between LPS and MIF. that MIF the of protein kinase has mechanism by which MIF may protein kinase acts as a of including which is important the activation of T. M. Calandra T. J. PubMed Scopus Google Scholar). the mechanism of between MIF and LPS, we have the functional link between MIF and the receptor which has been reported T. David J. M.P. Calandra T. PubMed Scopus Google Scholar), we to any regulatory link between MIF and at the protein in found no effect of MIF on of CD74 on cell that MIF may LPS and as a LPS on these we that MIF is a pro-inflammatory cytokine is inflammatory of LPS response to pro-inflammatory cytokine and to the activity of via of and role in the be to of the response and to of to and and and and of MIF, are to the with