Molecular Interaction and Enzymatic Activity of Macrophage Migration Inhibitory Factor with Immunorelevant Peptides
2003; Elsevier BV; Volume: 278; Issue: 33 Linguagem: Inglês
10.1074/jbc.m302854200
ISSN1083-351X
AutoresIlaria Potolicchio, Laura Santambrogio, Jack L. Strominger,
Tópico(s)Nuclear Receptors and Signaling
ResumoDisulfide reduction is an important step in antigen processing for HLA class II restricted T cell responses. Migration inhibitory factor (MIF) is a member of the thioredoxin family and has been classically defined as a cytokine. Using enzyme-linked immunosorbent assay and CD analysis, here we describe the binding to MIF of two peptides, hepatitis B surface antigen (HBsAg) and insulin B (InsB) with high affinity for HLA class II allo-types, HLA-DP2 and HLA-DQ8, respectively. At neutral pH, cysteinylated InsB was a substrate for MIF thiol reductase activity, as assessed by mass spectroscopy/electrospray analysis. Finally, a biologically active form of MIF co-immunopurified with mature forms of HLA DP2/15, and a peptide derived from the HLA-DP β1 helix could be used for affinity purification of MIF. The possibility that MIF participates in class II antigen presentation and/or as a chaperone is discussed. Disulfide reduction is an important step in antigen processing for HLA class II restricted T cell responses. Migration inhibitory factor (MIF) is a member of the thioredoxin family and has been classically defined as a cytokine. Using enzyme-linked immunosorbent assay and CD analysis, here we describe the binding to MIF of two peptides, hepatitis B surface antigen (HBsAg) and insulin B (InsB) with high affinity for HLA class II allo-types, HLA-DP2 and HLA-DQ8, respectively. At neutral pH, cysteinylated InsB was a substrate for MIF thiol reductase activity, as assessed by mass spectroscopy/electrospray analysis. Finally, a biologically active form of MIF co-immunopurified with mature forms of HLA DP2/15, and a peptide derived from the HLA-DP β1 helix could be used for affinity purification of MIF. The possibility that MIF participates in class II antigen presentation and/or as a chaperone is discussed. The macrophage migration inhibitory factor (MIF) 1The abbreviations used are: MIF, migration inhibitory factor; rMIF, recombinant MIF; HA, hemagglutinin; HBsAg, hepatitis B surface antigen; InsB, insulin B; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; LPS, lipopolysaccharide; TNF, tumor necrosis factor; Ab, antibody; Hsp, heat shock protein; ELISA, enzyme-linked immunosorbent assay; PBL, peripheral blood leukocytes.1The abbreviations used are: MIF, migration inhibitory factor; rMIF, recombinant MIF; HA, hemagglutinin; HBsAg, hepatitis B surface antigen; InsB, insulin B; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; LPS, lipopolysaccharide; TNF, tumor necrosis factor; Ab, antibody; Hsp, heat shock protein; ELISA, enzyme-linked immunosorbent assay; PBL, peripheral blood leukocytes. is a multifunctional protein involved in several inflammatory disorders (1Bernhagen J. Calandra T. Bucala R. J. Mol. Med. 1998; 76: 151-161Crossref PubMed Scopus (155) Google Scholar, 2Calandra T. Bucala R. Crit. Rev. Immunol. 1997; 17: 77-88Crossref PubMed Google Scholar), such as inflammatory lung diseases (3Donnelly S.C. Haslett C. Reid P.T. Grant I.S. Wallace W.A. Metz C.N. Bruce L.J. Bucala R. Nat. Med. 1997; 3: 320-323Crossref PubMed Scopus (392) Google Scholar), septic shock (4Calandra T. Echtenacher B. Roy D.L. Pugin J. Metz C.N. Hultner L. Heumann D. Mannel D. Bucala R. Glauser M.P. Nat. Med. 2000; 6: 164-170Crossref PubMed Scopus (688) Google Scholar), chronic colitis (5de Jong Y.P. Abadia-Molina A.C. Satoskar A.R. Clarke K. Rietdijk S.T. Faubion W.A. Mizoguchi E. Metz C.N. Alsahli M. ten Hove T. Keates A.C. Lubetsky J.B. Farrell R.J. Michetti P. van Deventer S.J. Lolis E. David J.R. Bhan A.K. Terhorst C. Sahli M.A. Nat. Immunol. 2001; 2: 1061-1066Crossref PubMed Scopus (269) Google Scholar), and some autoimmune diseases, e.g. rheumatoid arthritis (6Onodera S. Kaneda K. Mizue Y. Koyama Y. Fujinaga M. Nishihira J. J. Biol. Chem. 2000; 275: 444-450Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). MIF is widely expressed in different cell types but is produced mostly by activated macrophages and lymphocytes during inflammatory processes (7Lue H. Kleemann R. Calandra T. Roger T. Bernhagen J. Microbes Infect. 2002; 4: 449-460Crossref PubMed Scopus (302) Google Scholar). The full extent of its physiological role is not completely defined, although MIF has been implicated in several biological activities. First, MIF can function as a pro-inflammatory protein, as assessed by its inhibitory effect on dexamethasone-mediated TNF-α production (2Calandra T. Bucala R. Crit. Rev. Immunol. 1997; 17: 77-88Crossref PubMed Google Scholar) and its ability to sustain CD3-mediated T cell proliferation (8Bacher M. Metz C.N. Calandra T. Mayer K. Chesney J. Lohoff M. Gemsa D. Donnelly T. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7849-7854Crossref PubMed Scopus (612) Google Scholar) and pro-inflammatory cytokine secretion (9Calandra T. Bernhagen J. Metz C.N. Spiegel L.A. Bacher M. Donnelly T. Cerami A. Bucala R. Nature. 1995; 377: 68-71Crossref PubMed Scopus (1044) Google Scholar). MIF also possesses enzymatic activities as a tautomerase/isomerase (10Rosengren E. Bucala R. Aman P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar) and thiol oxidoreductase (11Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar). However, the natural substrates for MIF catalytic activity are unknown even though its enzymatic activity has been characterized in detail using insulin and l-DOPA as models (11Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar, 12Taylor A.B. Johnson Jr., W.H. Czerwinski R.M. Li H.S. Hackert M.L. Whitman C.P. Biochemistry. 1999; 38: 7444-7452Crossref PubMed Scopus (80) Google Scholar).X-ray crystallographic analysis of both human and rat recombinant MIF molecules showed a homotrimeric structure (13Sun H.W. Bernhagen J. Bucala R. Lolis E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5191-5196Crossref PubMed Scopus (287) Google Scholar, 14Suzuki M. Sugimoto H. Nakagawa A. Tanaka I. Nishihira J. Sakai M. Nat. Struct. Biol. 1996; 3: 259-266Crossref PubMed Scopus (187) Google Scholar). However, MIF is predominantly expressed as the monomer (44%) and dimer (33%), whereas only a smaller fraction (23%) is assembled to form a trimer (15Bendrat K. Al-Abed Y. Callaway D.J. Peng T. Calandra T. Metz C.N. Bucala R. Biochemistry. 1997; 36: 15356-15362Crossref PubMed Scopus (142) Google Scholar, 16Mischke R. Kleemann R. Brunner H. Bernhagen J. FEBS Lett. 1998; 427: 85-90Crossref PubMed Scopus (51) Google Scholar). Native MIF presumably possesses all three configurations of its recombinant forms. However, the relationship, if any, between configuration and biological activity is still to be defined.Antigen-presenting cells are able to internalize extracellular proteins within the acidic endosomal and lysosomal compartment to generate peptides for HLA class II loading. Antigen processing consists of protein denaturation and fragmentation. Several proteases have been described to be part of this system (17Villadangos J.A. Bryant R.A. Deussing J. Driessen C. Lennon-Dumenil A.M. Riese R.J. Roth W. Saftig P. Shi G.P. Chapman H.A. Peters C. Ploegh H.L. Immunol. Rev. 1999; 172: 109-120Crossref PubMed Scopus (204) Google Scholar). Proteins that contain disulfide bonds require an additional processing step, the reduction of the cysteine residues to produce antigenic peptides (18Collins D.S. Unanue E.R. Harding C.V. J. Immunol. 1991; 147: 4054-4059PubMed Google Scholar). Notably, modification of cysteine residues has been recently shown to regulate T cell responses to HLA class II-restricted epitopes (19Haque M.A. Hawes J.W. Blum J.S. J. Immunol. 2001; 166: 4543-4551Crossref PubMed Scopus (51) Google Scholar, 20Haque M.A. Li P. Jackson S.K. Zarour H.M. Hawes J.W. Phan U.T. Maric M. Cresswell P. Blum J.S. J. Exp. Med. 2002; 195: 1267-1277Crossref PubMed Scopus (110) Google Scholar, 21Jensen P.E. J. Exp. Med. 1991; 174: 1121-1130Crossref PubMed Scopus (89) Google Scholar). Within the endosomal pathway the γ-interferon-inducible lysosomal thiol reductase, the activity of which is restricted to acidic pH, has been found associated with antigen processing using reduction of cysteinylated peptides as the assay (22Maric M. Arunachalam B. Phan U.T. Dong C. Garrett W.S. Cannon K.S. Alfonso C. Karlsson L. Flavell R.A. Cresswell P. Science. 2001; 294: 1361-1365Crossref PubMed Scopus (218) Google Scholar).Antigenic peptides can also be generated extracellularly by secretion of several proteases from professional antigen-presenting cells or stressed cells, such as virally infected and tumor cells (23Santambrogio L. Sato A.K. Carven G.J. Belyanskaya S.L. Strominger J.L. Stern L.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15056-15061Crossref PubMed Scopus (162) Google Scholar, 24Monji T. Pious D. J. Immunol. 1997; 158: 3155-3164PubMed Google Scholar, 25Fox B.S. Carbone F.R. Germain R.N. Paterson Y. Schwartz R.H. Nature. 1988; 331: 538-540Crossref PubMed Scopus (48) Google Scholar). Additionally exogenous antigens or already unfolded proteins can bind directly to surface HLA class II molecule for T cell presentation (26Accapezzato D. Nisini R. Paroli M. Bruno G. Bonino F. Houghton M. Barnaba V. J. Immunol. 1998; 160: 5262-5266PubMed Google Scholar, 27Vacchino J.F. McConnell H.M. J. Immunol. 2001; 166: 6680-6685Crossref PubMed Scopus (16) Google Scholar).Here we describe the MIF peptide binding capacity and, more importantly, MIF thiol reductase activity at neutral pH on disulfide bonds in oxidized peptides. Because MIF is a secreted protein, particularly during inflammation, its thiol reductase activity could be associated with peptide modification in the extracellular milieu. Moreover, because MIF co-precipitated with HLA-DP2/15 molecules, a further role for MIF in HLA class II antigen presentation is suggested.EXPERIMENTAL PROCEDURESSynthetic Peptides—The following peptides were synthesized by Research Genetics (Waltham, MA): residues β 63–77 (KDILEEERAVPDRMA) of HLA-DP2 molecule; residues 306 –318 (PKYVKQNTLKLAT) of influenza virus hemagglutinin (HA); residues 14 –33 (VLQAGFFLLTRILTIPQSLD) of hepatitis B surface antigen (HBsAg); residues 85–99 (ENPVVHFFKNIVTPR) of myelin basic protein and residues 9 –23 (SHLVEALYLVCGERG) from the insulin B chain (InsB).Cloning and Bacterial Expression of Human MIF—MIF was amplified from the cDNA of a human B cell line, NP-1, and cloned in an isopropyl-1-thio-β-d-galactopyranoside-inducible expression vector (PGMT-7). PCR primers were designed as previously described with NdeI (5′-GCA TAT ACA TAT GCC GAT GTT CAT CGT A-3′) and BamHI (5′-TTG GAT CCT TAG GCG AAG GTG GAG TT-3′). MIF-PGMT-7 was expressed in Escherichia coli BL21-(DE3) pLysS+ cells (Invitrogen). One liter of bacterial cell culture was grown at 37 °C until the optical density at 595 nm reached was 0.7–1 absorbance units, and then the cell culture was incubated with 1 mm isopropyl-1-thio-β-d-galactopyranoside for2hat30 °C. The pellet was resuspended in 5 ml/g BugBaster protein extraction Reagent (Novagen, Madison, WI) for 20–45 min in the presence of DNase and protease inhibitors. Cell debris were removed by centrifugation, and after filtration through 0.45-μm filters, the supernatant was applied to Mono Q column (Amersham Biosciences) and an overnight dialysis at 4 °Cin20mm Tris, pH 7.5, 20 mm NaCl. Because MIF is not retained by Mono Q resin, the flow-through was collected, and MIF was purified by gel filtration chromatography (Superdex 200, Pharmacia).ELISA and CD Spectrum Analysis—ELISA plates were coated overnight with 50 μm each peptide and then blocked for 1 h in 2% bovine serum albumin, phosphate-buffered saline at room temperature. Two micrograms of recombinant MIF (rMIF) were added and incubated overnight with the peptides. After washing, bound rMIF was detected in DuoSet ELISA (R&D, Minneapolis, MN). For CD spectrum analysis 25 μm rMIF was incubated overnight at room temperature with or without 2.5 μm each peptide. The incubation was performed in HSB (20 mm Hepes, 0.85% NaCl). The next day samples were diluted 10× in water, concentrated with MICRON10 (Millipore, Bedford, MA), and analyzed on a Jasco CD spectrometer. Secondary structure was analyzed on line by using the k2d program (www.emblheidelberg.de/~andrade/k2d).Thiol Reductase Activity of MIF—Insulin B, residues 9 –23, was oxidized as previously described. Briefly, peptide was incubated with 400 μm Cys-Hank's balanced salt solution for 3 h and then dialyzed for 48 h against 10 mm Hepes, pH 7.0. 0.5 μg of oxidized peptide was incubated for 20 min with increasing amounts of rMIF, 100, 200, and 500 ng in 10 μl of 0.4 mm Cys-10 mm Hepes buffer, pH 7.0. Samples were purified by ZipTip (Millipore) and analyzed by mass spectrometry electrospray. Mass spectrometry electrospray analysis was carried out at the Chemistry Department at Harvard University.Immunoaffinity Columns—HLA-DRB1*0401 was purified from 10 g of B cell line pellet (PRIESS). HLA-DP2/15 was purified from 20 g of a heterozygous B cell line (NB-1) transformed and previously typed. Pellets were resuspended in lysis buffer containing 50 mm Tris-HCl, pH 7.5, 0.1 mm NaCl, complete EDTA-free protease inhibitory mixture tablets (Roche Applied Science), and either 1% Nonidet P-40 or 0.5% CHAPS. After centrifugation protein extract was filtered through a 0.2-μm filter and put through a series of columns: POROS(r)20 AL-NMS, POROS(r)20 A, POROS(r)20 AL-LB3.1, POROS(r)20 AL-IVD12, and POROS(r)20 AL-B7/21 as previously described (35Malik P. Strominger J.L. J. Immunol. Methods. 2000; 234: 83-88Crossref PubMed Scopus (13) Google Scholar). After a protocol set up in this laboratory, immunoaffinity columns were washed with solution I (0.1% Nonidet P-40, 50 mm Tris-HCl, pH 8.0), solution II (0.5% CHAPS, 500 mm NaCl, 50 mm Tris, pH 8.8), and solution III (0.1% CHAPS, 2 mm Tris-HCl, pH 8.0). Elution was in 0.1% CHAPS, 50 mm glycine buffer, pH 11.5. Buffer exchange and protein concentration were performed on Centricon 10. Small scale protein purification was obtained using cell lysate from 5 g of B cell line. Native MIF was isolated using the monoclonal antibody anti-human MIF (R&D) conjugated to N-hydroxysuccinimide-activated resin (Amersham Biosciences). Second-step purification was performed using either B7/21 alone or a mix of HLA class II monoclonal antibodies, B7/21 and IVD12, conjugated to N-hydroxysuccinimide-activated column. PRIESS and NB-1 were both analyzed. Proteins were run on 12.5% SDS gels and analyzed by Coomassie Blue staining or Western blot. HLA class II isotypes were stained with a general anti-HLA class II rabbit serum, generously provided by Dr. N. Tanigaki. Human MIF was stained using polyclonal biotinylated antibody specific for human MIF (R&D).Analysis of the Inhibitory Effect of MIF on Dexamethasone Activity— MIF function was tested as previously described. Briefly, human monocytes were isolated from whole blood by Ficoll density gradient centrifugation and plated at 5 × 106 cells/ml in 24-well plates in RPMI, 10% human AB serum. The monocytes were purified by adherence and incubated for 1 h with dexamethasone (10–9m) plus 1–10 ng/ml of rMIF (R&D) or 1 ng of human MIF co-purified with HLA-DP2/15 from B7/21 column. LPS (Sigma) was added at the concentration of 0.5 μg/ml. Sixteen hours of conditioned medium was collected, and TNF-α secretion was quantified by ELISA (DuoSet R&D).Pulse-Chase and Immunoprecipitation—LB and Bg5 B cells lines were pulsed for 30 min with 1 μCi/ml [35S]methionine (PerkinElmer Life Sciences) and then chased for 1 and 4 h in complete Dulbecco's modified Eagle's medium supplemented with a 10-fold excess of cold methionine and cysteine. Cells were lysed in 1% Nonidet P-40 150 mm NaCl in 50 mm Tris containing a mixture of protease inhibitors (Roche Applied Science) and equivalent amounts of radioactive post-nuclear supernatant used for immunoprecipitation after preclearing with protein A- and G-Sepharose beads (Sigma). The immunoprecipitation was performed using a mouse anti-human MIF mAb (clone 12302.2) (R&D system). Samples were resolved by SDS-PAGE boiled and non-boiled under non-reducing conditions.Peptide Affinity Column—A HiTrap N-hydroxysuccinimide-activated 1-ml column (Amersham Biosciences) was conjugated with 5 mg of solubilized peptide following the protocol provided by the company. Five grams of a transformed human B cell line were lysed in 25 mm Tris-HCl, 0.5 mm NaCl, 0.5% CHAPS, and complete, EDTA-free, protease inhibitor mixture tablets (Roche Applied Science). The affinity column was loaded overnight at 4 °C with cell lysate and then extensively washed in lysis buffer. Elution was completed with 100 mm glycine buffer, pH 3. Fractions corresponding to the elution peak were concentrated on Micron 10 (Millipore) and analyzed by Coomassie staining of 12.5% SDS-PAGE. The whole elution was trypsin-digested and sequenced by matrix-assisted laser desorption ionization mass spectroscopy analysis. Tandem mass spectroscopy analysis was carried out at the Microchemistry Facility at Harvard University.RESULTSMIF Peptide Binding Analysis—Macrophage migration inhibitory factor functions as a tautomerase/isomerase and thiol reductase on small molecule substrates such as l-DOPA and insulin (11Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar, 12Taylor A.B. Johnson Jr., W.H. Czerwinski R.M. Li H.S. Hackert M.L. Whitman C.P. Biochemistry. 1999; 38: 7444-7452Crossref PubMed Scopus (80) Google Scholar). Pro-1 and Cys-57–Cys-60 form the enzymatic catalytic sites. Thioreductase activity has an optimal pH ranging between 7.3 and 9 (11Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar). MIF is a soluble protein mostly secreted extracellularly. Because a number of different molecules with enzymatic properties have been found involved in extracellular protein fragmentation and processing (23Santambrogio L. Sato A.K. Carven G.J. Belyanskaya S.L. Strominger J.L. Stern L.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15056-15061Crossref PubMed Scopus (162) Google Scholar, 24Monji T. Pious D. J. Immunol. 1997; 158: 3155-3164PubMed Google Scholar, 25Fox B.S. Carbone F.R. Germain R.N. Paterson Y. Schwartz R.H. Nature. 1988; 331: 538-540Crossref PubMed Scopus (48) Google Scholar) the possible interaction between MIF and immunologically relevant peptides was examined. Human rMIF was expressed and purified from E. coli BL21-(DE3) Lys+ cells as previously described (28Suzuki M. Murata E. Tanaka I. Nishihira J. Sakai M. J. Mol. Biol. 1994; 235: 1141-1143Crossref PubMed Scopus (25) Google Scholar). Correct protein folding assessed by CD spectrum analysis (see Fig. 2) was identical to that previously published (29Mischke R. Gessner A. Kapurniotu A. Juttner S. Kleemann R. Brunner H. Bernhagen J. FEBS Lett. 1997; 414: 226-232Crossref PubMed Scopus (33) Google Scholar). Several well known high affinity HLA class II peptides were selected and analyzed for their ability to interact with rMIF in an ELISA assay (Fig. 1) (30Chicz R.M. Urban R.G. Lane W.S. Gorga J.C. Stern L.J. Vignali D.A. Strominger J.L. Nature. 1992; 358: 764-768Crossref PubMed Scopus (670) Google Scholar). The highest amount of rMIF was detected in plates coated with HBsAg14–33, a peptide derived from hepatitis B surface antigen, which showed a high binding affinity for HLA-DP2 (31Chicz R.M. Graziano D.F. Trucco M. Strominger J.L. Gorga J.C. J. Immunol. 1997; 159: 4935-4942PubMed Google Scholar). A fragment of insulin B (InsB9–23) known to bind HLA-DQ8 (32Lee K.H. Wucherpfennig K.W. Wiley D.C. Nat. Immunol. 2001; 2: 501-507Crossref PubMed Scopus (321) Google Scholar) also showed a significant binding capacity to rMIF, whereas little or no binding was observed for both myelin basic protein residues 85–99 and HA306–318, peptides, known for their high affinity binding to HLA-DR2 and HLA-DR1, respectively (33O'Sullivan D. Sidney J. Appella E. Walker L. Phillips L. Colon S.M. Miles C. Chesnut R.W. Sette A. J. Immunol. 1990; 145: 1799-1808PubMed Google Scholar). Although ELISA is a well established method to determine HLA peptide binding, a better approach to examine protein-protein interaction is analyzing conformational changes by CD spectrum analysis. In agreement with what has been previously reported, the rMIF CD spectrum showed a positive ellipticity at 197 nm and broad negative ellipticity between 205 and 225 nm (Fig. 2) (29Mischke R. Gessner A. Kapurniotu A. Juttner S. Kleemann R. Brunner H. Bernhagen J. FEBS Lett. 1997; 414: 226-232Crossref PubMed Scopus (33) Google Scholar). Under the conditions used, the secondary structure obtained with the K2d program was 37% α helix, 15% β sheet, and 48% random coil. Moreover, changes in the MIF CD spectrum were observed after an overnight incubation with HBsAg14–33 peptide (Fig. 2a). Secondary structure analysis by K2d revealed a consistent increase of β sheets and decrease of random coils, 41 and 31%, respectively. No modifications, either in CD spectrum or in secondary structure, were observed after an overnight incubation with HA306–318 or myelin basic protein residues 85–99 peptides (Fig. 2b). At the concentration used all of the peptides had only a very weak signal that coincided with the noise level (Fig. 2c). These observations suggest that rMIF has a flexible conformation, and major modifications of the secondary structure may occur upon complex formation with specific peptides.Fig. 1Analysis of peptide binding MIF. rMIF was added to ELISA plates previously coated with 50 μm HLA class II naturally processed peptides. The amount of bound protein was quantified using a biotinylated polyclonal antibody specific for human MIF followed by horseradish peroxidase-conjugated-Streptavidin. Absorbance was measured at 450 nm. The data shown represent a typical experiment where each sample is assayed in triplicate. The experiment was repeated three times.View Large Image Figure ViewerDownload Hi-res image Download (PPT)MIF Thiol Reductase Activity—MIF enzymatic activity includes a thiol reductase function (11Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar). Site-directed mutation analysis pointed to Cys-57 and Cys-60 as part of the MIF enzymatic catalytic site, determined in vitro using molecular models (11Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar). Further analysis was conducted here to investigate whether the MIF thiol reductase activity could function on oxidized peptides. Among the synthetic peptides tested for their binding to rMIF (Fig. 1), InsB9–23, molecular mass 1645.8, possesses a cysteine residue in position 11. Cysteinylation of this residue in Cys-Hank's balanced salt solution changed the molecular mass to 1765, as shown by mass spectrometry electrospray analysis (Fig. 3, a–b). Peaks at 823 and at 883 are double-charged and correspond to m/z of reduced and oxidized peptide, respectively. After 20 min of incubation with rMIF at pH 7.2 a significant amount of InsB9–23 was reduced (Fig. 3c). The ratio peptide reduced/oxidized was dose-dependent as shown in Fig. 3d. Thus, rMIF can function directly to reduce cysteinylated forms of InsB 9–23. As expected, no modifications in the molecular mass of the cysteinylated peptide occurred without adding rMIF (Fig. 3b). Thus, peptides can be substrates of MIF thiol reductase, and as expected, this reaction occurs at neutral pHFig. 3MIF thiol reductase activity. Mass spectrometry electrospray analysis of reduced InsB9–23, m+2 823 (a), oxidized InsB9–23, m+2 882 (b), and oxidized InsB 9–23 after incubation with 200 ng of rMIF (c). d, schematic representation of the dose-dependent reduction of Cys-InsB9–23 by purified rMIF. The ratio of relative peaks, m+2 reduce peptide and m+2 cysteinylated peptide, are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)MIF and HLA Class II Association—A possible association between MIF and HLA class II molecules was also assessed. Recently a modified methodology based on high pressure immunoaffinity columns that is suitable for rapid large scale purification of HLA proteins was described (34Gorga J.C. Horejsi V. Johnson D.R. Raghupathy R. Strominger J.L. J. Biol. Chem. 1987; 262: 16087-16094Abstract Full Text PDF PubMed Google Scholar, 35Malik P. Strominger J.L. J. Immunol. Methods. 2000; 234: 83-88Crossref PubMed Scopus (13) Google Scholar). This technique was applied to purify HLA-DR (HLA-DRB1*0401) using mAb LB3.1 and HLA DP (HLA-DPB1*0201/*1501) using mAb B7/21 affinity columns. The isolated HLA molecules were analyzed on Western blot using a general HLA class II rabbit antiserum as the detection antibody (Fig. 4a, left). Interestingly, MIF was found associated with HLA-DP2/15 mainly as the dimer, as detected by Western blot analysis, but not with the HLA-DR4 preparation (Fig. 4a, right).Fig. 4Analysis of human MIF interaction with HLA class II molecules. a, Western blot analysis of immunoaffinity-purified HLADR4 and HLA-DP2/15 molecules and Western blot analysis of MIF co-precipitated with HLA class II molecules. MIF monomeric and dimeric forms are indicated. b, quantification of the amount of MIF found in association with HLA-DP2/15 molecules (filled diamonds) and HLA-DR4 (filled circle). Protein standards are indicated as open squares. c, secretion of TNF-α in untreated PBLs (Unt, lane 1) and in LPSstimulated PBLs (lane 2). The inhibitory effect of dexamethasone (Dex) on TNF-α secretion in LPS stimulated PBLs in shown in lane 3. The antagonist effect of rMIF (1–10 ng/ml) (lane 4) and human MIF (hMIF, 1 ng/ml) co-isolated with HLA-DP2/15 (lane 5) on TNF-α secretion in dexamethasone-LPS treated PBLs are shown. Secretion of TNF-α in PBLs is shown treated with dexamethasone (lane 6), rMIF (lane 7), or human MIF (lane 8) without LPS stimulation. NB, non-boiled.View Large Image Figure ViewerDownload Hi-res image Download (PPT)MIF has been characterized by its ability to override the inhibitory effect of dexamethasone on TNF-α production from LPS-stimulated human peripheral blood mononuclear cells (9Calandra T. Bernhagen J. Metz C.N. Spiegel L.A. Bacher M. Donnelly T. Cerami A. Bucala R. Nature. 1995; 377: 68-71Crossref PubMed Scopus (1044) Google Scholar). This assay was, therefore, used to assess the biological activity of MIF found in association with HLA-DP2/15. First, sandwich ELISA was performed to detect the MIF concentration in the HLA-DP preparation (Fig. 4b). The HLA-DR preparation was also analyzed, and as expected, no MIF was found associated. As shown in Fig. 4c, MIF that had been co-purified with HLA-DP2/15 as well as purified recombinant MIF partially antagonized the inhibitory effect of dexamethasone on TNF-α secretion to an extent similar to that previously reported (9Calandra T. Bernhagen J. Metz C.N. Spiegel L.A. Bacher M. Donnelly T. Cerami A. Bucala R. Nature. 1995; 377: 68-71Crossref PubMed Scopus (1044) Google Scholar).Pulse-chase experiments were performed to investigate whether MIF could be found associated with HLA DP2/15 at any step of class II trafficking from the endoplasmic reticulum to the cell surface. HLA class II heterodimers were chased up to 18 h; however, MIF could not be identified after precipitation of HLA-DP (data not shown). This result was not surprising since, besides the invariant chain, which associates with all nascent class II α/β heterodimers, all other proteins known to pair with HLA class II molecules (e.g. HLA-DM, HLA-DO (36Brocke P. Garbi N. Momburg F. Hammerling G.J. Curr. Opin. Immunol. 2002; 14: 22-29Crossref PubMed Scopus (70) Google Scholar), tetraspanin (37Engering A. Pieters J. Int. Immunol. 2001; 13: 127-134Crossref PubMed Scopus (83) Google Scholar, 38Kropshofer H. Spindeldreher S. Rohn T.A. Platania N. Grygar C. Daniel N. Wolpl A. Langen H. Horejsi V. Vogt A.B. Nat. Immunol. 2002; 3: 61-68Crossref PubMed Scopus (194) Google Scholar), and CD1d (39Kang S.J. Cresswell P. EMBO J. 2002; 21: 1650-1660Crossref PubMed Scopus (108) Google Scholar)) only interact with a small percentage of the class II α/β complexes. HLA-DP-associated MIF was detected by first immunopurifying with the mAb specific for human MIF (Fig. 5a) followed by a second immunopurification step using mAb B7–21 specific for HLA-DP molecules (Fig. 5b). Interestingly, MIF is associated with peptide-loaded forms of HLA-DP, as indicated by the presence of an SDS stable class II α/β complex. In these experimental conditions, MIF dimers and monomers were purified using both anti-DP and anti-MIF column (Figs. 4a, 5a, and 6c) and the dimeric form of MIF became monomeric under reducing conditions (Fig. 5a). In pulse-chase experiments on two different B cell lines (LB, Bg5),
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