Macrophage Migration Inhibitory Factor Promotes Intestinal Tumorigenesis
2005; Elsevier BV; Volume: 129; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2005.07.061
ISSN1528-0012
AutoresJonathan M. Wilson, P. Louise Coletta, Richard Cuthbert, Nigel Scott, Kenneth MacLennan, Gillian Hawcroft, Lin Leng, Jodi B. Lubetsky, Kai K. Jin, Elias Lolis, Francisco J. Medina, José A. Brieva, Richard Poulsom, Alexander F. Markham, Richard Bucala, Mark A. Hull,
Tópico(s)Nuclear Receptors and Signaling
ResumoBackground & Aims: The cytokine macrophage migration inhibitory factor (MIF) is expressed throughout the human gastrointestinal tract. Recently, protumorigenic activity of MIF has been described in several cancer models. Therefore, we investigated the expression and function of MIF during the early stages of intestinal tumorigenesis. Methods: MIF messenger RNA, protein, and tautomerase activity were measured in normal intestinal mucosa and adenomas from patients with sporadic colorectal adenomas and in the adenomatous polyposis coli (Apc)Min/+ mouse model of intestinal tumorigenesis. MIF function was investigated by using VACO-235 human colorectal adenoma cells in vitro and by testing the effect of genetic deletion of Mif on ApcMin/+ mouse intestinal tumorigenesis. Results: MIF expression and tautomerase activity were increased in human and ApcMin/+ mouse intestinal adenomas compared with adjacent normal mucosa. Up-regulation of MIF occurred mainly in epithelial cells (associated with an increasing grade of dysplasia), but also in stromal plasma cells. Exogenous MIF inhibited apoptosis and promoted anchorage-independent growth of VACO-235 cells (maximal at 100 ng/mL). Homozygous deletion of Mif was associated with a reduction in the number and size of ApcMin/+ mouse adenomas (P = .025 for the difference in large [>7-mm] tumors) and decreased angiogenesis (43% decrease in mean tumor microvessel density), but there was no alteration in epithelial cell apoptosis or proliferation. Conclusions: MIF expression is increased in sporadic human colorectal adenomas, and exogenous MIF drives tumorigenic behavior of epithelial cells in vitro. Mif also promotes intestinal tumorigenesis (predominantly via angiogenesis) in the ApcMin/+ mouse. Therefore, MIF is a potential colorectal cancer chemoprevention target. Background & Aims: The cytokine macrophage migration inhibitory factor (MIF) is expressed throughout the human gastrointestinal tract. Recently, protumorigenic activity of MIF has been described in several cancer models. Therefore, we investigated the expression and function of MIF during the early stages of intestinal tumorigenesis. Methods: MIF messenger RNA, protein, and tautomerase activity were measured in normal intestinal mucosa and adenomas from patients with sporadic colorectal adenomas and in the adenomatous polyposis coli (Apc)Min/+ mouse model of intestinal tumorigenesis. MIF function was investigated by using VACO-235 human colorectal adenoma cells in vitro and by testing the effect of genetic deletion of Mif on ApcMin/+ mouse intestinal tumorigenesis. Results: MIF expression and tautomerase activity were increased in human and ApcMin/+ mouse intestinal adenomas compared with adjacent normal mucosa. Up-regulation of MIF occurred mainly in epithelial cells (associated with an increasing grade of dysplasia), but also in stromal plasma cells. Exogenous MIF inhibited apoptosis and promoted anchorage-independent growth of VACO-235 cells (maximal at 100 ng/mL). Homozygous deletion of Mif was associated with a reduction in the number and size of ApcMin/+ mouse adenomas (P = .025 for the difference in large [>7-mm] tumors) and decreased angiogenesis (43% decrease in mean tumor microvessel density), but there was no alteration in epithelial cell apoptosis or proliferation. Conclusions: MIF expression is increased in sporadic human colorectal adenomas, and exogenous MIF drives tumorigenic behavior of epithelial cells in vitro. Mif also promotes intestinal tumorigenesis (predominantly via angiogenesis) in the ApcMin/+ mouse. Therefore, MIF is a potential colorectal cancer chemoprevention target. The proinflammatory cytokine macrophage migration inhibitory factor (MIF) plays a critical role in host innate and acquired immunity.1Calandra T. Roger T. Macrophage migration inhibitory factor a regulator of innate immunity.Nat Rev Immunol. 2003; 3: 791-800Crossref PubMed Scopus (1289) Google Scholar Multiple functions have been ascribed to MIF, including activation of macrophages and T cells and antagonism of endogenous glucocorticoid activity.1Calandra T. Roger T. Macrophage migration inhibitory factor a regulator of innate immunity.Nat Rev Immunol. 2003; 3: 791-800Crossref PubMed Scopus (1289) Google Scholar MIF has been implicated in the pathogenesis of several acute and chronic inflammatory conditions, including septic shock and arthritis.1Calandra T. Roger T. Macrophage migration inhibitory factor a regulator of innate immunity.Nat Rev Immunol. 2003; 3: 791-800Crossref PubMed Scopus (1289) Google Scholar As would be expected of a critical regulator of the early innate host response to pathogens, MIF protein is expressed constitutively in the human gastrointestinal tract, primarily by epithelial cells, but also by a poorly characterized lamina propria cell population.2Maaser C. Eckmann L. Paesold G. Kim H.S. Kagnoff M.F. Ubiquitous production of macrophage migration inhibitory factor by human gastric and intestinal epithelium.Gastroenterology. 2002; 122: 667-680Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar Increased plasma MIF levels are recognized in ulcerative colitis and Crohn’s disease patients.3Murakami H. Fazle Akbar S.K.M. Matsui H. Onji M. Macrophage migration inhibitory factor in the sera and at the colonic mucosa in patients with ulcerative colitis clinical implications and pathogenic significance.Eur J Clin Invest. 2001; 31: 337-343Crossref PubMed Scopus (77) Google Scholar, 4de Jong Y.P. Abadia-Molina A.C. Satoskar A.R. Clarke K. Rietdijk S.T. Faubion W.A. Mizoguchi E. Metz C.N. Al Sahli 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. Development of chronic colitis is dependent on the cytokine MIF.Nat Immunol. 2001; 2: 1061-1066Crossref PubMed Scopus (269) Google Scholar Moreover, genetic deletion of Mif or antibody-mediated neutralization of Mif protein abrogates the development of experimental colitis in rodents.4de Jong Y.P. Abadia-Molina A.C. Satoskar A.R. Clarke K. Rietdijk S.T. Faubion W.A. Mizoguchi E. Metz C.N. Al Sahli 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. Development of chronic colitis is dependent on the cytokine MIF.Nat Immunol. 2001; 2: 1061-1066Crossref PubMed Scopus (269) Google Scholar, 5Ohkawara T. Nishihira J. Takeda H. Hige S. Kato M. Sugiyama T. Iwanaga T. Nakamura H. Mizue Y. Asaka M. Amelioration of dextran sulfate sodium-induced colitis by anti-macrophage migration inhibitory factor antibody in mice.Gastroenterology. 2002; 123: 256-270Abstract Full Text Full Text PDF PubMed Scopus (136) Google ScholarMore recently, MIF has been implicated in carcinogenesis in a variety of in vitro and in vivo models.6Mitchell R.A. Mechanisms and effectors of MIF-dependent promotion of tumourigenesis.Cell Signal. 2004; 16: 13-19Crossref PubMed Scopus (122) Google Scholar, 7Nishihira J. Ishibashi T. Fukushima T. Sun B. Sato Y. Todo S. Macrophage migration inhibitory factor (MIF) its potential role in tumor growth and tumor-associated angiogenesis.Ann N Y Acad Sci. 2003; 995: 171-182Crossref PubMed Scopus (115) Google Scholar MIF abrogates p53-dependent apoptosis of macrophages and promotes RAS-mediated transformation of fibroblasts.8Hudson J.D. Shoaibi M.A. Maestro R. Carnero A. Hannon G.J. Beach D.H. A proinflammatory cytokine inhibits p53 tumor suppressor activity.J Exp Med. 1999; 190: 1375-1382Crossref PubMed Scopus (566) Google Scholar, 9Fingerle-Rowson G. Petrenko O. Metz C.N. Forsthuber T.G. Mitchell R. Huss R. Moll U. Muller W. Bucala R. The p53-dependent effects of macrophage migration inhibitory factor revealed by gene targeting.Proc Natl Acad Sci U S A. 2003; 100: 9354-9359Crossref PubMed Scopus (240) Google Scholar, 10Petrenko O. Fingerle-Rowson G. Peng T. Mitchell R.A. Metz C.N. Macrophage migration inhibitory factor deficiency is associated with altered cell growth and reduced susceptibility to Ras-mediated transformation.J Biol Chem. 2003; 278: 11078-11085Crossref PubMed Scopus (56) Google Scholar MIF has also been implicated in lymphoma and melanoma cell tumor growth and angiogenesis in rodents.11Chesney J. Metz C. Bacher M. Peng T. Meinhardt A. Bucala R. An essential role for macrophage migration inhibitory factor (MIF) in angiogenesis and the growth of a murine lymphoma.Mol Med. 1999; 5: 181-191Crossref PubMed Google Scholar, 12Shimizu T. Abe R. Nakamura H. Ohkawara A. Suzuki M. Nishihira J. High expression of macrophage migration inhibitory factor in human melanoma cells and its role in tumor cell growth and angiogenesis.Biochem Biophys Res Commun. 1999; 264: 751-758Crossref PubMed Scopus (186) Google Scholar Therefore, MIF may be added to the increasing list of proinflammatory cytokines (eg, tumor necrosis factor α), chemokines (eg, CXCL12), and transcription factors (eg, nuclear factor-κ B [NF-κB]) now implicated in carcinogenesis.13Robinson S.C. Coussens L.M. Soluble mediators of inflammation during tumor development.Adv Cancer Res. 2005; 93: 159-187Crossref PubMed Scopus (98) Google Scholar, 14Balkwill F. Cancer and the chemokine network.Nat Rev Cancer. 2004; 4: 540-550Crossref PubMed Scopus (1884) Google Scholar, 15Greten F.R. Eckmann L. Greten T.F. Park J.M. Li Z.-W. Egan L.J. Kagnoff M.F. Karin M. IKKβ links inflammation and tumorigenesis in a mouse model of colitis-associated cancer.Cell. 2004; 118: 285-296Abstract Full Text Full Text PDF PubMed Scopus (2065) Google ScholarOverexpression of MIF has been shown in several human neoplasms,6Mitchell R.A. Mechanisms and effectors of MIF-dependent promotion of tumourigenesis.Cell Signal. 2004; 16: 13-19Crossref PubMed Scopus (122) Google Scholar including prostate,16del Vecchio M.T. Tripodi S.A. Arcuri F. Pergola L. Hako L. Vatti R. Cintorino M. Macrophage migration inhibitory factor in prostatic adenocarcinoma correlation with tumor grading and combination endocrine treatment-related changes.Prostate. 2000; 45: 51-57Crossref PubMed Scopus (68) Google Scholar breast,17Bando H. Matsumoto G. Bando M. Muta M. Ogawa T. Funata N. Nishihira J. Koike M. Toi M. Expression of macrophage migration inhibitory factor in human breast cancer association with nodal spread.Jpn J Cancer Res. 2002; 93: 389-396Crossref PubMed Scopus (102) Google Scholar and lung18Kamimura A. Kamachi M. Nishihira J. Ogura S. Isobe H. Dosaka-Akita H. Ogata A. Shindoh M. Ohbuchi T. Kawakami Y. Intracellular distribution of macrophage migration inhibitory factor predicts prognosis of patients with adenocarcinoma of the lung.Cancer. 2000; 89: 334-341Crossref PubMed Scopus (139) Google Scholar cancer. One study, predating cloning and isolation of human MIF, showed that macrophage migration inhibitory activity was present in 85% of sporadic colorectal cancer (CRC) extracts and 50% of colorectal adenoma extracts, unlike normal colorectal tissue extracts, which were consistently negative.19Shkolnik T. Livni E. Reshef R. Lachter J. Eidelman S. The macrophage migration inhibition (MIF) assay as a marker of colorectal cancer. Studies in patients with colorectal cancer, noncolonic neoplasms, and conditions predisposing to colorectal cancer.Dis Colon Rectum. 1987; 30: 101-105Crossref PubMed Scopus (10) Google Scholar More recently, increased MIF protein levels have been shown in human sporadic CRC tissue compared with normal colorectal mucosa in 1 immunohistochemical study,20Legendre H. Decaestecker C. Nagy N. Hendlisz A. Schuring M.-P. Salmon I. Gabius H.-J. Pector J.-C. Kiss R. Prognostic values of galectin-3 and the macrophage migration inhibitory factor (MIF) in human colorectal cancers.Mod Pathol. 2003; 16: 491-504Crossref PubMed Scopus (87) Google Scholar and MIF expression has been detected in several human and mouse CRC cell lines, including HT29, CaCo2, LoVo, and colon 26.2Maaser C. Eckmann L. Paesold G. Kim H.S. Kagnoff M.F. Ubiquitous production of macrophage migration inhibitory factor by human gastric and intestinal epithelium.Gastroenterology. 2002; 122: 667-680Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 7Nishihira J. Ishibashi T. Fukushima T. Sun B. Sato Y. Todo S. Macrophage migration inhibitory factor (MIF) its potential role in tumor growth and tumor-associated angiogenesis.Ann N Y Acad Sci. 2003; 995: 171-182Crossref PubMed Scopus (115) Google Scholar, 20Legendre H. Decaestecker C. Nagy N. Hendlisz A. Schuring M.-P. Salmon I. Gabius H.-J. Pector J.-C. Kiss R. Prognostic values of galectin-3 and the macrophage migration inhibitory factor (MIF) in human colorectal cancers.Mod Pathol. 2003; 16: 491-504Crossref PubMed Scopus (87) Google Scholar In the last cell line, RNA interference and antibody-mediated neutralization of Mif have both been shown to impair tumor growth and neovascularization in syngeneic BALB/c mice.7Nishihira J. Ishibashi T. Fukushima T. Sun B. Sato Y. Todo S. Macrophage migration inhibitory factor (MIF) its potential role in tumor growth and tumor-associated angiogenesis.Ann N Y Acad Sci. 2003; 995: 171-182Crossref PubMed Scopus (115) Google Scholar, 21Sun B. Nishihira J. Suzuki M. Fukushima N. Ishibashi T. Kondo M. Sato Y. Todo S. Induction of macrophage migration inhibitory factor by lysophosphatidic acid relevance to tumor growth and angiogenesis.Int J Mol Med. 2003; 12: 633-641PubMed Google ScholarHowever, a role for MIF during the early stages of intestinal tumorigenesis, before transition from the premalignant, benign colorectal adenoma (or polyp) to an invasive adenocarcinoma or cancer, has not yet been studied. Because these stages are relevant to the development of CRC chemoprevention strategies, we addressed the hypothesis that MIF promotes the early stages of sporadic (as opposed to inflammatory bowel disease–associated) colorectal carcinogenesis. To this end, we investigated MIF expression in human sporadic colorectal adenomas and in intestinal adenomas from the adenomatous polyposis coli (Apc)Min/+ mouse model of familial adenomatous polyposis. We also tested the effect of exogenous MIF on human colorectal adenoma cells in vitro and the effect of genetic deletion of Mif on ApcMin/+ mouse intestinal tumorigenesis in vivo.Materials and MethodsHuman TissueApproval for the use of archival and fresh human colorectal tissue, as well as clinicopathologic data, was obtained from the St James’s and Seacroft University Hospitals Local Research Ethics Committee. Formalin-fixed, paraffin-embedded (FFPE) specimens of human sporadic colorectal adenomas were randomly selected from the Histopathology Department archives at St James’s University Hospital. Tissue from patients with inflammatory bowel disease or familial adenomatous polyposis was not analyzed.Fresh colorectal adenoma tissue and paired macroscopically normal colorectal mucosa (6 biopsy samples taken at least 2 cm away from the adenoma) were obtained by endoscopic polypectomy and biopsy. Most of each adenoma was placed in 10% (vol/vol) formalin under the supervision of a consultant histopathologist. The remaining adenoma material and paired normal colorectal mucosa were washed separately in 0.9% (wt/vol) saline and placed immediately into ice-cold phosphate-buffered saline (PBS), pH 7.4 (Sigma, Poole, UK), containing Protease Inhibitor Cocktail III (Calbiochem, San Diego, CA). Samples were then homogenized on ice by using an ULTRA-TURAX T8 homogenizer (Ika-Labortech, Dortmund, Germany) and then sonicated (10-μm amplitude for 10 seconds) 3 times (Sanyo SONIPREP 150; Sanyo Loughborough, UK) before centrifugation at 7000g for 20 minutes at 4°C. The supernatant was aspirated and used for total protein estimation by Bio-RadDC protein assay (Hercules, CA) and assay of tautomerase activity (see below). Other fresh colorectal adenoma tissue was embedded in OCT compound (Merck Ltd, Poole, UK) and snap-frozen in isopentane cooled in liquid nitrogen before storage at −70°C.The following clinicopathologic parameters were recorded: age and sex of the patient, adenoma size (maximum diameter in millimeters for polypectomy specimens), position (distal or proximal to the splenic flexure), histological type (tubular, tubulovillous, or villous), and the highest grade of dysplasia (mild, moderate, or severe).Animal StudiesC57BL/6 × 129/Sv Mif−/− mice22Bozza M. Satoskar A.R. Lin G. Lu B. Humbles A.A. Gerard C. David J.R. Targeted disruption of migration inhibitory factor gene reveals its critical role in sepsis.J Exp Med. 1999; 189: 341-346Crossref PubMed Scopus (491) Google Scholar (a kind gift from Dr John David, Boston, MA) were mated with C57BL/6 ApcMin/+ animals (The Jackson Laboratory, Bar Harbor, ME) in specific pathogen–free conditions, as described previously.23Scott D.J. Hull M.A. Cartwright E.J. Lam W.K. Tisbury A. Poulsom R. Markham A.F. Bonifer C. Coletta P.L. Lack of inducible nitric oxide synthase promotes intestinal tumorigenesis in the ApcMin/+ mouse.Gastroenterology. 2001; 121: 889-899Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar Allele-specific polymerase chain reaction (PCR) genotype analysis was performed as described previously.22Bozza M. Satoskar A.R. Lin G. Lu B. Humbles A.A. Gerard C. David J.R. Targeted disruption of migration inhibitory factor gene reveals its critical role in sepsis.J Exp Med. 1999; 189: 341-346Crossref PubMed Scopus (491) Google Scholar, 23Scott D.J. Hull M.A. Cartwright E.J. Lam W.K. Tisbury A. Poulsom R. Markham A.F. Bonifer C. Coletta P.L. Lack of inducible nitric oxide synthase promotes intestinal tumorigenesis in the ApcMin/+ mouse.Gastroenterology. 2001; 121: 889-899Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar Comparison of the intestinal phenotype of C57BL/6 × 129/Sv Mif−/− × ApcMin/+ vs Mif+/+ × ApcMin/+ mice of both sexes was performed between 106 and 124 days of age by a single observer blinded to the Mif genotype. Immediately after death, the entire gastrointestinal tract from the duodenum to the rectum was removed, opened longitudinally, and washed thoroughly with PBS. The number and size (maximum diameter in millimeters) of all adenomas in the small intestine (SI) and colon were measured separately by using a Lynx stereo dissecting microscope fitted with a graticule.23Scott D.J. Hull M.A. Cartwright E.J. Lam W.K. Tisbury A. Poulsom R. Markham A.F. Bonifer C. Coletta P.L. Lack of inducible nitric oxide synthase promotes intestinal tumorigenesis in the ApcMin/+ mouse.Gastroenterology. 2001; 121: 889-899Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar Intestinal tissue was fixed in 4% (wt/vol) paraformaldehyde (Sigma) in PBS at 25°C overnight before storage in 70% (vol/vol) ethanol and subsequent embedding in paraffin. Tissue sections were stained with H&E and underwent histological examination by an experienced histopathologist, who was blinded to the origin of each section. Homogenate supernatants of normal intestinal mucosa and adenomas were obtained as described previously.Apoptotic Epithelial Cell Counting in ApcMin/+ Mouse Intestinal AdenomasIn ApcMin/+ mouse intestinal adenomas, 2 patterns of distribution of apoptotic bodies can be discerned, namely, scattered apoptosis within the epithelial layer and crypt lumen apoptosis.24Brodie C.M. Crotty P.L. Gaffney E.F. Morphologically distinct patterns of apoptosis correlate with size and high-grade dysplasia in colonic adenomas.Histopathology. 2004; 44: 240-246Crossref PubMed Scopus (6) Google Scholar In human colorectal adenomas, the degree of crypt lumen apoptosis is associated with a larger tumor size and increasing dysplasia.24Brodie C.M. Crotty P.L. Gaffney E.F. Morphologically distinct patterns of apoptosis correlate with size and high-grade dysplasia in colonic adenomas.Histopathology. 2004; 44: 240-246Crossref PubMed Scopus (6) Google Scholar Therefore, both the number of scattered intraepithelial apoptotic bodies (defined as cells with nuclear chromatin condensation, nuclear fragmentation, and condensed, eosinophilic cytoplasm surrounded by a clear halo) per 1000 epithelial cells (scattered apoptosis) and the number of crypt lumina that contained apoptotic bodies in 20 randomly selected crypts (crypt lumen apoptosis) were counted by 1 blinded observer in all the ApcMin/+ mouse intestinal adenomas examined.Recombinant Human MIFEndotoxin-free (<0.2 EU/mg) recombinant human (rh) MIF in Tris-buffered saline (TBS; pH 7.4) was synthesized as described previously.25Lubetsky J.B. Swope M. Dealwis C. Blake P. Lolis E. Pro-1 of macrophage migration inhibitory factor functions as a catalytic base in the phenylpyruvate tautomerase activity.Biochemistry. 1999; 38: 7346-7354Crossref PubMed Scopus (130) Google Scholar, 26Lubetsky J.B. Dios A. Han J. Aljabari B. Ruzsicska B. Mitchell R. Lolis E. Al-Abed Y. The tautomerase active site of macrophage migration inhibitory factor is a potential target for discovery of novel anti-inflammatory agents.J Biol Chem. 2002; 277: 24976-24982Crossref PubMed Scopus (232) Google ScholarAssay for p-Hydroxyphenylpyruvate Tautomerase ActivityMeasurement of p-hydroxyphenylpyruvate (HPP) tautomerase activity was performed as described previously.25Lubetsky J.B. Swope M. Dealwis C. Blake P. Lolis E. Pro-1 of macrophage migration inhibitory factor functions as a catalytic base in the phenylpyruvate tautomerase activity.Biochemistry. 1999; 38: 7346-7354Crossref PubMed Scopus (130) Google Scholar Briefly, HPP was dissolved in 50 mmol/L ammonium acetate (pH 6.0) to a concentration of 100 mmol/L and stored at 4°C. Tissue or cell lysate (200 μL) or rhMIF was added to a quartz cuvette containing 10 μL of 100 mmol/L HPP in 300 μL of 0.435 mol/L boric acid (pH 6.2) and 690 μL of distilled water at 25°C. The increase in optical density (OD) at 330 nm between 10 and 60 seconds at 25°C was measured (ΔOD33010–60 s), and the value was corrected for the background ΔOD33010–60 s value in the presence of PBS only. The HPP tautomerase activity of tissue or cell homogenate supernatants was expressed as ΔOD33010–60 s per milligram of total protein.The presence of protease inhibitors in the lysis buffer did not affect HPP tautomerase activity in colorectal mucosal homogenates (data not shown). Intra-assay and interassay coefficients of variation for the HPP tautomerase assay (using 1.23 μg of rhMIF) were 3.6% (n = 15) and 6.8% (n = 20) respectively.Enzyme-Linked Immunoassay for MIFMIF protein levels were quantified in cell culture media or cell/tissue homogenate supernatants by using a human MIF enzyme-linked immunoassay (EIA; Chemicon International Inc, Temecula, CA) according to the manufacturer’s instructions. All samples and rhMIF standards were tested in duplicate. Results are expressed as nanograms of MIF per milligrams of total protein.Western Blot AnalysisProtein homogenates (10 μg) or rhMIF was mixed with an equal volume of 2× Laemmli sample buffer (125 mmol/L Tris-HCl [pH 6.9], 2% [wt/vol] sodium dodecyl sulfate, 700 μmol/L β-mercaptoethanol, 10% [vol/vol] glycerol, and 0.1% [vol/vol] bromophenol blue). Samples were heated to 70°C for 10 minutes before sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weight markers (Sea Blue Plus 2; Invitrogen Ltd, Paisley, UK) by using a Bio-Rad Mini-Protean II apparatus. Resolved proteins were transferred onto a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, UK) by wet transfer. Membranes were incubated with blocking buffer (0.02% [vol/vol] Tween-20 [Sigma] and 5% [wt/vol] nonfat dry milk in PBS) for 16 hours at 4°C and then washed (3 × 2 minutes) in 0.05% (vol/vol) Tween-20 in PBS (wash buffer) at 25°C. Mouse monoclonal anti-human MIF antibody (R&D Systems Europe Ltd, Abingdon, UK) was diluted 1:500 with blocking buffer and incubated with membranes for 2 hours at 25°C. Mouse monoclonal anti-human β-actin antibody (Sigma) was diluted 1:2500 in blocking buffer and incubated with membranes for 1 hour at 25°C. After washing (3 × 2 minutes), membranes were incubated with a 1:5000 dilution of horseradish peroxidase–conjugated rabbit anti-mouse antibody (Sigma) in blocking buffer for 1 hour at 25°C. After further washes (3 × 2 minutes), proteins were detected by using enhanced chemiluminescence (Pierce, IL).ImmunohistochemistryFFPE sections (3 μm thick) were mounted on Superfrost Plus slides (BDH, Poole, UK). Sections were dewaxed in xylene and rehydrated through a graded alcohol series. Endogenous peroxidase activity was blocked with 0.6% (vol/vol) H2O2 in 100% methanol for 15 minutes at 25°C. After washing in water for 10 minutes, sections underwent antigen retrieval by microwave heating for 10 minutes at 100°C in 10 mmol/L citrate buffer (pH 6.0), followed by cooling in tap water and an avidin/biotin block (Avidin/Biotin Blocking kit; Vector Laboratories, Burlingame, CA). Nonspecific binding sites were blocked with 10% (vol/vol) rabbit serum (DakoCytomation Ltd, Ely, UK) in TBS (50 mmol/L Tris and 0.15 mol/L NaCl, pH 7.4) for 30 minutes at 25°C. Mouse monoclonal anti-human MIF antibody (1 mg/mL; R&D Systems) was incubated with sections for 2 hours at 25°C. After washing in TBS (2 × 5 minutes), sections were incubated with biotinylated rabbit anti-mouse immunoglobulin (Ig)G (DakoCytomation Ltd; 1:200 dilution in 10% [vol/vol] rabbit serum) for 30 minutes at 25°C. After further washes with TBS (2 × 5 minutes), sections were incubated with streptavidin/biotin-horseradish peroxidase complex (DakoCytomation Ltd) for 30 minutes at 25°C. Sections were washed in TBS (2 × 5 minutes) and then visualized by using 3,3′-diaminobenzidine tetrahydrochloride (0.45 mg/mL) and 0.01% (vol/vol) hydrogen peroxide in 0.2 mol/L Tris-HCl (pH 7.6) for 10 minutes at 25°C. Sections were counterstained in Mayer’s hematoxylin for 3 minutes, dehydrated by using a sequential alcohol and xylene series, and mounted in diphenylxylene. Negative controls used included omission of the primary antibody, incubation with 1 μg/mL irrelevant isotype control mouse IgG1 (Sigma), and preadsorption of the primary antibody with 10 μg/mL rhMIF (R&D Systems) overnight at 4°C, before incubation with sections.Immunohistochemistry for Mif on fixed sections of mouse intestine was performed as described previously except that the primary antibody was an affinity-purified goat polyclonal anti-human MIF antibody (5 μg/mL; R&D Systems) and biotinylated rabbit anti-goat IgG was used as a secondary antibody. This primary antibody was also used for immunohistochemistry for MIF on human colorectal adenomas.Immunohistochemistry for CD138, CD68, and CD3 was performed as described previously except that antibodies to CD138 (Serotec Ltd, Oxford, UK), CD68 (Dakocytomation Ltd), and CD3 (DakoCytomation Ltd) were incubated with sections for 1 hour at 25°C at a 1:100 dilution with no prior avidin/biotin blocking step. Sections for CD3 staining required trypsin antigen retrieval (0.1% [wt/vol] trypsin II-S [Sigma] in 0.1% CaCl2 [pH 7.8] for 60 seconds at 37°C).Immunofluorescence for MIF was performed on 5-μm frozen sections of unfixed human adenoma tissue by blocking with 10% (vol/vol) normal goat serum (DakoCytomation Ltd) for 60 minutes at 25°C and using mouse monoclonal anti-MIF antibody (1 μg/mL) overnight at 4°C. After 2 washes with TBS/0.02% (vol/vol) Tween-20 (5 minutes each), sections were fixed in 100% acetone at −20°C for 10 minutes and then air-dried. Sections were then incubated with Cy3-conjugated affinity-purified goat anti-mouse IgG (Jackson Immunoresearch Laboratories, PA) at a dilution of 1:400 in 10% (vol/vol) goat serum for 30 minutes at 25°C. After further washes, sections were mounted in Vectashield (Vector Laboratories). Controls for immunofluorescence included omission of the primary antibody and preadsorption of the primary antibody by rhMIF, as described previously.Semiquantitative Analysis of MIF and CD138 ImmunostainingMIF staining of FFPE sections of human colorectal adenomas was assessed by 2 observers blinded to the origin and clinicopathologic characteristics of each section. A consensus score of the combined intensity and distribution of MIF staining in epithelial cells and in stromal cells was produced (0, no staining; 1, weak, patchy staining; 2, moderate or strong staining of patchy distribution; 3, strong, uniform staining) for each adenoma.The significance of differences in MIF epithelial and stromal scores of adenomas related to clinicopathologic factors was tested with the Mann–Whitney U test or Kruskal–Wallis analysis of variance. Logistic regression with stepwise selection was performed to identify independent factors from those identified by univariate analysis that predicted increased MIF immunoreactivity. The mean number of MIF-positive and CD138-positive stromal and lamina propria cells per microscopic high-power field (400× magnification) was determined from 5 high-power fields of sections from each of 9 human colorectal adenomas and 10 samples of histologically normal (HN) colorectal mucosa by a single observer blinded to the stromal score assigned to each adenoma.Determination of the Microvessel Density and Proliferation Index of ApcMin/+ Mouse Intestinal AdenomasMicrovessel density (MVD) in ApcMin/+ mouse intestinal adenomas was measured as the number of von Willebrand factor–positive structures in 1 low-power (100× magnification) field of each adenoma, counted at 400× magnification by a single observer blinded to the tumor genotype as described previously.27Seno H. Oshima M. Ishikawa T. Oshima H. Takaku K. Chiba T. Narumiya S. Taketo M.M. Cyclooxygenase 2- and prostaglandin E(2) receptor EP(2)-dependent angiogenesis in Apc(Delta716) mouse intestinal polyps.Cancer Res. 2002; 62: 506-511PubMed Google Scholar Immunohistochemistry was performed on 4% (wt/vol) paraformaldehyde-fixed paraffin-embedded intestinal tissue as described previously, except that rabbit polyclonal
Referência(s)