Title: Functional Expression of the Interleukin-11 Receptor α-Chain and Evidence of Antiapoptotic Effects in Human Colonic Epithelial Cells
Abstract: A tissue-protective effect of interleukin-11 (IL-11) for the intestinal mucosa has been postulated from animal models of inflammatory bowel disease (IBD). Despite the fact that the clinical usefulness of the anti-inflammatory effects of this cytokine is presently investigated in patients with IBD, there are no data available regarding the target cells of IL-11 action and the mechanisms of tissue protection within the human colonic mucosa. IL-11 responsiveness is restricted to cells that express the interleukin-11 receptor α-chain (IL-11Rα) and an additional signal-transducing subunit (gp130). In this study, we identified the target cells for IL-11 within the human colon with a new IL-11Rα monoclonal antibody and investigated the functional expression of the receptor and downstream effects of IL-11-induced signaling. Immunohistochemistry revealed expression of the IL-11Rα selectively on colonic epithelial cells. HT-29 and colonic epithelial cells (CEC) constitutively expressed IL-11Rα mRNA and protein. Co-expression of the signal-transducing subunit gp130 was also demonstrated. IL-11 induced signaling through triggering activation of the Jak-STAT pathway without inducing anti-inflammatory or proliferative effects in colonic epithelial cells. However, IL-11 stimulation resulted in a dose-dependent tyrosine phosphorylation of Akt, a decreased activation of caspase-9, and a reduced induction of apoptosis in cultured CEC. In HLA-B27 transgenic rats treated with IL-11, a reduction of apoptotic cell numbers was found. This study demonstrates functional expression of the IL-11Rα restricted on CEC within the human colonic mucosa. IL-11 induced signaling through triggering activation of the Jak-STAT pathway, without inducing anti-inflammatory or proliferative effects. The beneficial effects of IL-11 therapy are likely to be mediated by CEC via activation of the Akt-survival pathway, mediating antiapoptotic effects to support mucosal integrity. A tissue-protective effect of interleukin-11 (IL-11) for the intestinal mucosa has been postulated from animal models of inflammatory bowel disease (IBD). Despite the fact that the clinical usefulness of the anti-inflammatory effects of this cytokine is presently investigated in patients with IBD, there are no data available regarding the target cells of IL-11 action and the mechanisms of tissue protection within the human colonic mucosa. IL-11 responsiveness is restricted to cells that express the interleukin-11 receptor α-chain (IL-11Rα) and an additional signal-transducing subunit (gp130). In this study, we identified the target cells for IL-11 within the human colon with a new IL-11Rα monoclonal antibody and investigated the functional expression of the receptor and downstream effects of IL-11-induced signaling. Immunohistochemistry revealed expression of the IL-11Rα selectively on colonic epithelial cells. HT-29 and colonic epithelial cells (CEC) constitutively expressed IL-11Rα mRNA and protein. Co-expression of the signal-transducing subunit gp130 was also demonstrated. IL-11 induced signaling through triggering activation of the Jak-STAT pathway without inducing anti-inflammatory or proliferative effects in colonic epithelial cells. However, IL-11 stimulation resulted in a dose-dependent tyrosine phosphorylation of Akt, a decreased activation of caspase-9, and a reduced induction of apoptosis in cultured CEC. In HLA-B27 transgenic rats treated with IL-11, a reduction of apoptotic cell numbers was found. This study demonstrates functional expression of the IL-11Rα restricted on CEC within the human colonic mucosa. IL-11 induced signaling through triggering activation of the Jak-STAT pathway, without inducing anti-inflammatory or proliferative effects. The beneficial effects of IL-11 therapy are likely to be mediated by CEC via activation of the Akt-survival pathway, mediating antiapoptotic effects to support mucosal integrity. Interleukin-11 (IL-11) 1The abbreviations used are: IL, interleukin; IL-11Rα, interleukin-11 receptor α-chain; Ab, antibody; CEC, human primary colonic epithelial cells; FITC, fluorescein isothiocyanate; G3PDH, glyceraldehyde-3-phosphate-dehydrogenase; IBD, inflammatory bowel disease; Jak, Janus kinase; mAb, monoclonal antibody; PE, phycoerythrin; STAT, signal transducer and activator of transcription; TNF, tumor necrosis factor; PBS, phosphate-buffered saline; FCS, fetal calf serum; PI, propidium iodine; RT, reverse transcriptase; TUNEL, terminal dUTP nick-end labeling. was initially cloned as a mediator of plasmacytoma cell proliferation (1Paul S.R. Bennett F. Calvetti J.A. Kelleher K. Wood C.R. O'Hara Jr., R.M. Leary A.C. Sibley B. Clark S.C. Williams D.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7512-7516Crossref PubMed Scopus (572) Google Scholar) and was later found to exhibit a wide variety of biological effects in neural cells as well as in the hematopoietic and the immune system (for a review, see Ref. 2Du X. Williams D.A. Blood. 1997; 89: 3897-3908Crossref PubMed Google Scholar). IL-11 is a member of a family of cytokines that includes interleukin-6 (IL-6), leukemia inhibitory factor, oncostatin M, and ciliary neurotropic factor (3Zhang X.G. Gu J.J. Lu Z.Y. Yasukawa K. Yancopoulos G.D. Turner K. Shoyab M. Taga T. Kishimoto T. Bataille R. J. Exp. Med. 1994; 179: 1337-1342Crossref PubMed Scopus (227) Google Scholar, 4Yin T. Taga T. Tsang M.L. Yasukawa K. Kishimoto T. Yang Y.C. J. Immunol. 1993; 151: 2555-2561PubMed Google Scholar). These so-called IL-6-type cytokines drive the assembly of a multisubunit receptor complex that initiates intracellular signaling by association with the transmembrane signal transducer glycoprotein gp130 (5Fourcin M. Chevalier S. Lebrun J.J. Kelly P. Pouplard A. Wijdenes J. Gascan H. Eur. J. Immunol. 1994; 24: 277-280Crossref PubMed Scopus (50) Google Scholar, 6Dahmen H. Horsten U. Kuster A. Jacques Y. Minvielle S. Kerr I.M. Ciliberto G. Paonessa G. Heinrich P.C. Muller-Newen G. Biochem. J. 1998; 331: 695-702Crossref PubMed Scopus (70) Google Scholar, 7Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1316) Google Scholar, 8Heinrich P.C. Behrmann I. Muller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1785) Google Scholar). IL-11 is specifically bound to a unique interleukin-11 receptor α-chain (IL-11Rα) (9Nandurkar H.H. Hilton D.J. Nathan P. Willson T. Nicola N. Begley C.G. Oncogene. 1996; 12: 585-593PubMed Google Scholar, 10Yang Y.C. Yin T. Ann. N. Y. Acad. Sci. 1995; 762: 31-40Crossref PubMed Scopus (32) Google Scholar). The human IL-11Rα was initially cloned from a bone marrow cDNA library and shares 85% nucleotide and 84% amino acid identity with the murine IL-11Rα, which was first cloned in 1994 (9Nandurkar H.H. Hilton D.J. Nathan P. Willson T. Nicola N. Begley C.G. Oncogene. 1996; 12: 585-593PubMed Google Scholar, 11Cherel M. Sorel M. Lebeau B. Dubois S. Moreau J.F. Bataille R. Minvielle S. Jacques Y. Blood. 1995; 86: 2534-2540Crossref PubMed Google Scholar, 12Hilton D.J. Hilton A.A. Raicevic A. Rakar S. Harrison-Smith M. Gough N.M. Begley C.G. Metcalf D. Nicola N.A. Willson T.A. EMBO J. 1994; 13: 4765-4775Crossref PubMed Scopus (256) Google Scholar). Recent studies show that IL-11-induced signaling is mediated by the formation of a hexameric receptor complex, composed of two molecules each of IL-11, IL-11Rα, and gp130 (13Barton V.A. Hall M.A. Hudson K.R. Heath J.K. J. Biol. Chem. 2000; 275: 36197-36203Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). An important signaling system activated by the IL-11Rα and other members of this receptor family is the Janus kinase-signal transducer and activator of transcription (Jak-STAT) pathway (8Heinrich P.C. Behrmann I. Muller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1785) Google Scholar, 14Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1271) Google Scholar). Specific receptor-binding of IL-11 triggers dimerization of gp130 and transient activation via transphosphorylation of tyrosine kinases of the Jak family. IL-11 has been shown to induce activation of Jak1 and Jak2 receptor kinases downstream of IL-11Rα/gp130 activation (15Yin T. Yasukawa K. Taga T. Kishimoto T. Yang Y.C. Exp. Hematol. 1994; 22: 467-472PubMed Google Scholar, 16Berger L.C. Hawley T.S. Lust J.A. Goldman S.J. Hawley R.G. Biochem. Biophys. Res. Commun. 1994; 202: 596-605Crossref PubMed Scopus (48) Google Scholar). Jaks phosphorylate tyrosine residues in the cytoplasmic regions of gp130, which in turn serve as docking site for STAT1 and STAT3 proteins (17Stahl N. Farruggella T.J. Boulton T.G. Zhong Z. Darnell J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1353Crossref PubMed Scopus (876) Google Scholar). These signaling molecules are members of the STAT family of transcription factors (18Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5157) Google Scholar, 19Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3448) Google Scholar). Recruited STAT proteins undergo tyrosine phosphorylation and dissociate from gp130, dimerize (20Zhong Z. Wen Z. Darnell J.E. Science. 1994; 264: 95-98Crossref PubMed Scopus (1772) Google Scholar), and translocate into the nucleus, where they act as transcriptional activators of cytokine-responsive genes (14Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1271) Google Scholar, 21Akira S. Int. J. Biochem. Cell Biol. 1997; 29: 1401-1418Crossref PubMed Scopus (199) Google Scholar). IL-11 has been shown to be an important mediator in sytems other than the hematopoietic system (22Du X.X. Williams D.A. Blood. 1994; 83: 2023-2030Crossref PubMed Google Scholar). IL-11 reduces the expression of a number of proinflammatory cytokines in several cell culture systems. Preclinical in vivo and in vitro studies have demonstrated that IL-11 inhibits the secretion of tumor necrosis factor (TNF), interleukin-12 (IL-12), interleukin-1β (IL-1β), and nitric oxide from activated macrophages as well as interferon-γ and IL-12 from activated T cells (23Leng S.X. Elias J.A. J. Immunol. 1997; 159: 2161-2168PubMed Google Scholar, 24Opal S.M. Keith J.C. Palardy J.E. Parejo N. J. Infect. Dis. 2000; 181: 754-756Crossref PubMed Scopus (12) Google Scholar, 25Trepicchio W.L. Ozawa M. Walters I.B. Kikuchi T. Gilleaudeau P. Bliss J.L. Schwertschlag U. Dorner A.J. Krueger J.G. J. Clin. Invest. 1999; 104: 1527-1537Crossref PubMed Scopus (210) Google Scholar, 26Trepicchio W.L. Bozza M. Pedneault G. Dorner A.J. J. Immunol. 1996; 157: 3627-3634PubMed Google Scholar). In addition, IL-11 exerts cytoprotective effects on the intestinal mucosa (27Schwertschlag U.S. Trepicchio W.L. Dykstra K.H. Keith J.C. Turner K.J. Dorner A.J. Leukemia. 1999; 13: 1307-1315Crossref PubMed Scopus (156) Google Scholar, 28Peterson R.L. Bozza M.M. Dorner A.J. Am. J. Pathol. 1996; 149: 895-902PubMed Google Scholar, 29Orazi A. Du X. Yang Z. Kashai M. Williams D.A. Lab. Invest. 1996; 75: 33-42PubMed Google Scholar, 30Potten C.S. Stem Cells. 1996; 14: 452-459Crossref PubMed Scopus (83) Google Scholar). IL-11 treatment reduced mucosal damage after chemotherapy and radiation (31Du X.X. Doerschuk C.M. Orazi A. Williams D.A. Blood. 1994; 83: 33-37Crossref PubMed Google Scholar, 32Du X. Liu Q. Yang Z. Orazi A. Rescorla F.J. Grosfeld J.L. Williams D.A. Am. J. Physiol. 1997; 272: G545-G552Crossref PubMed Google Scholar, 33Qiu B.S. Pfeiffer C.J. Keith J.C. Dig. Dis. Sci. 1996; 41: 1625-1630Crossref PubMed Scopus (110) Google Scholar). Remarkably, IL-11 ameliorated disease severity in colitis models, such as HLA B-27 transgenic rats (34Peterson R.L. Wang L. Albert L. Keith Jr., J.C. Dorner A.J. Lab. Invest. 1998; 78: 1503-1512PubMed Google Scholar, 35Keith Jr., J.C. Albert L. Sonis S.T. Pfeiffer C.J. Schaub R.G. Stem Cells. 1994; 12: 79-90PubMed Google Scholar, 36Greenwood-Van Meerveld B. Tyler K. Keith Jr., J.C. Lab. Invest. 2000; 80: 1269-1280Crossref PubMed Scopus (44) Google Scholar) and attenuated cell death after intestinal ischemia-reperfusion (37Kuenzler K.A. Pearson P.Y. Schwartz M.Z. J. Surg. Res. 2002; 108: 268-272Abstract Full Text PDF PubMed Scopus (30) Google Scholar). Because of its therapeutic effectiveness in several animal models, IL-11 was considered to be a promising drug candidate for the treatment of inflammatory bowel diseases (IBD). Recently, Sands et al. (38Sands B.E. Winston B.D. Salzberg B. Safdi M. Barish C. Wruble L. Wilkins L. Shapiro M. Schwertschlag U.S. the RHIL-11 Crohn's Study GroupAliment. Pharmacol. Ther. 2002; 16: 399-406Crossref PubMed Scopus (139) Google Scholar) in a randomized, controlled trial revealed that treatment with recombinant human interleukin-11 in patients with active Crohn's disease was well tolerated. However, there was only a trend to a decreased Crohn's disease activity index in the recombinant human IL-11 group versus placebo. Despite the fact that the clinical utility of the anti-inflammatory properties and beneficial effects of this cytokine are presently being investigated in clinical studies in patients with Crohn's disease (27Schwertschlag U.S. Trepicchio W.L. Dykstra K.H. Keith J.C. Turner K.J. Dorner A.J. Leukemia. 1999; 13: 1307-1315Crossref PubMed Scopus (156) Google Scholar, 39Grosfeld J.L. Du X. Williams D.A. JPEN J. Parenter. Enteral. Nutr. 1999; 23: S67-S69Crossref PubMed Scopus (21) Google Scholar, 40Sands B.E. Bank S. Sninsky C.A. Robinson M. Katz S. Singleton J.W. Miner P.B. Safdi M.A. Galandiuk S. Hanauer S.B. Varilek G.W. Buchman A.L. Rodgers V.D. Salzberg B. Cai B. Loewy J. DeBruin M.F. Rogge H. Shapiro M. Schwertschlag U.S. Gastroenterology. 1999; 117: 58-64Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar), there are no data regarding the target cells of IL-11 action and the mechanisms of tissue protection within the human colonic mucosa. IL-11 responsiveness is restricted to cells that express both the IL-11Rα subunit and the transmembrane signaling receptor gp130 on their surface (9Nandurkar H.H. Hilton D.J. Nathan P. Willson T. Nicola N. Begley C.G. Oncogene. 1996; 12: 585-593PubMed Google Scholar, 12Hilton D.J. Hilton A.A. Raicevic A. Rakar S. Harrison-Smith M. Gough N.M. Begley C.G. Metcalf D. Nicola N.A. Willson T.A. EMBO J. 1994; 13: 4765-4775Crossref PubMed Scopus (256) Google Scholar). Expression of gp130 protein has previously been described on many cells types and tissues (41Kishimoto T. Stem Cells. 1994; 12: 37-44PubMed Google Scholar); however, only preliminary data are available for the expression of IL-11Rα mRNA in murine intestinal cell lines (28Peterson R.L. Bozza M.M. Dorner A.J. Am. J. Pathol. 1996; 149: 895-902PubMed Google Scholar) and murine tissue (42Davidson A.J. Freeman S.A. Crosier K.E. Wood C.R. Crosier P.S. Stem Cells. 1997; 15: 119-124Crossref PubMed Scopus (40) Google Scholar). A recently generated IL-11Rα mAb (clone E24.2) reacting with an epitope expressed in the extracellular region of human IL-11Rα (43Blanc C. Vusio P. Schleinkofer K. Boisteau O. Pflanz S. Minvielle S. Grotzinger J. Muller-Newen G. Heinrich P.C. Jacques Y. Montero-Julian F.A. J. Immunol. Methods. 2000; 241: 43-59Crossref PubMed Scopus (10) Google Scholar) enabled us to investigate the expression of the IL-11Rα in mucosal tissue. A functional role for IL-11 and for its receptor moiety in the human colonic mucosa has not yet been established. In order to address these questions, we investigated the expression of IL-11Rα mRNA and protein in the colon cancer cell line HT-29 and in human primary colonic epithelial cells (CEC). We further analyzed signaling via tyrosine phosphorylation of downstream molecules upon treatment with IL-11 and possible effector mechanisms. In this study, colonic epithelial cells are shown to be the prime target of IL-11 in the human mucosa, since the functional surface expression of the receptor in conjunction with the gp130 protein could be demonstrated. Furthermore, our data provide evidence that IL-11 mediates activation of the Jak-STAT pathway within colonic epithelial cells, causing antiapoptotic but not anti-inflammatory or proliferative effects. In vivo studies in HLA-B27 transgenic rats revealed a reduction of apoptotic CEC in IL-11-treated animals. Preparation of Human Primary Colonic Epithelial Cells—Colonic tissue was obtained from patients undergoing surgical resection for colorectal carcinoma. The study was approved by the University of Regensburg Ethics Committee. Normal mucosa was taken at least 10 cm distant from the tumor. The mucosa was stripped from the submucosa within 30 min after bowel resection and rinsed several times with PBS at room temperature. The mucus was removed by incubating the specimens twice in 1 mm dithiothreitol (Sigma) for 15 min at 37 °C. After washing with PBS, the mucosa was rotated in 2 mm EDTA in Hanks' balanced salt solution without calcium and magnesium (PAA, Linz, Austria) for 10 min at 37 °C. The supernatant was discarded. The remaining mucosa was vortexed in PBS, and the supernatant containing complete crypts and some single cells was collected into a 15-ml tube. Vortexing was repeated until the supernatant was almost clear. To separate CEC-containing crypts from contaminating single cells, the suspension was allowed to settle down for up to 5 min at room temperature. The sedimented crypts were collected, washed with PBS, and resuspended in minimal essential medium supplemented with Earle salts, 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml gentamycin, 2.5 μg/ml fungizone (JRH Bioscience, Lenexa, KS). Media were purchased from Biochrom (Berlin, Germany), and supplements were obtained from Sigma. Cell Culture and Stimulation Conditions—HT-29 were obtained from American Type Culture Collection (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium (Biochrom) supplemented with 10% FCS, 1% nonessential amino acids, and 1% sodium pyruvate (Biochrom) under standard tissue culture conditions (10% CO2 at 37 °C). CEC were cultured on collagen A-coated nylon filters according to the method described earlier (44Rogler G. Daig R. Aschenbrenner E. Vogl D. Schlottmann K. Falk W. Gross V. Scholmerich J. Andus T. Lab. Invest. 1998; 78: 889-890PubMed Google Scholar). For the detection of tyrosine phosphorylation of Jak1 and STAT3 upon treatment with IL-11, HT-29 cells were cultured in Dulbecco's modified Eagle's medium containing 0.5% FCS for 48 h to reduce basal levels of intracellular phosphorylation. Subsequently, the medium was removed, and cells were incubated in medium without serum for 2 h and then treated with serum-free medium containing various concentrations of recombinant IL-11 (Genetics Institute, Cambridge, MA) for different periods of time. Since CEC rapidly undergo apoptosis after loss of anchorage from the mucosa (45Grossmann J. Walther K. Artinger M. Kiessling S. Scholmerich J. Cell Growth Differ. 2001; 12: 147-155PubMed Google Scholar), freshly EDTA-isolated crypts were immediately resuspended in minimal essential medium without FCS and centrifuged (3 min at 1200 rpm) to regain cell-cell contact. Thereafter, sedimented CEC were starved in serum-free minimal essential media for 2 h at 37 °C, resuspended in serum-free medium containing various concentrations of IL-11, and finally incubated at 37 °C for 15 min. For the investigation of antiapoptotic properties of IL-11 in CEC, cells were pretreated with different concentrations of IL-11 and kept in suspension by shaking to induce detachment-induced apoptosis. RT-PCR and Northern Blot Analysis—Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and reverse transcribed into first-strand cDNA. Primers specific for the human IL-11Rα (forward, 5′-CGTGAAGCTGTGTTGTCCTG-3′; reverse, 5′-GCTCCTAGGACTGTCTTCTTC-3′) and the human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as internal control (forward, 5′-TTAGCACCCCTGGCCAAGG-3′; reverse, 5′-CTTACTCCTTGGAGGCCATG-3′) were used for PCR (HOTstar; Qiagen). The sizes of the PCR products are 339 bp for IL-11Rα and 529 bp for G3PDH. PCR conditions for both were as follows: 95 °C for 15 min for denaturation with HOTstart polymerase, 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min for 35 cycles for IL-11Rα and 25 cycles for G3PDH, followed by 72 °C for 10 min. For Northern blot analysis, 15 μg of total RNA were size-fractionated on a 1.0% formaldehyde-agarose gel, transferred to a nylon membrane (Hybond™; Amersham Biosciences) and UV-cross-linked in a UV Stratalinker 1800 (Stratagene, Amsterdam, The Netherlands). The amplified cDNA probes encoding the human IL-11Rα and GAPDH were labeled with [32P]dATP (3000 Ci/mmol) (Amersham Biosciences) using a random primed DNA labeling kit (Roche Applied Science). The membranes were hybridized with the labeled probes for 1 h at 65 °C in Quikhyb solution (Stratagene). After extensive washing, the membranes were exposed to Kodak AR X-Omat films (Eastman Kodak Co.) at –80 °C using an intensifying screen. Flow Cytometric Analysis—To obtain a single cell suspension from the isolated crypts, CEC were incubated in dispase (1.2 mg/ml in Hanks' balanced salt solution; Roche Applied Science) for 2 min at 37 °C followed by vigorous shaking. Enzyme activity was stopped by the addition of EDTA (1 mm), and cells were fixed in 70% methanol at –20 °C until analysis. HT-29 cells were detached from culture flasks with accutase (Sigma), washed with PBS, pelleted at 4000 rpm, and resuspended in PBS plus 2% FCS. Following blocking for 30 min on ice, cells were incubated with specific anti-IL-11Rα mAb E24.2, anti-gp130 Ab (Upstate Biotechnology, Eching, Germany), or isotope control (mouse IgG1; Sigma) for 1 h on ice. Thereafter, cells were washed twice with PBS containing 2% FCS and stained with a secondary, PE-labeled rabbit anti-mouse Ab (DAKO, Hamburg, Germany) for 1 h. Samples were washed twice with PBS and resuspended in 500 μl of PBS for fluorescent analysis. For phenotypic staining, cells were incubated on ice for 1 h with FITC-labeled mouse anti-epithelial antigen mAb EP-4 (clone Ber-EP-4; DAKO) or IgG1 FITC (Coulter Immunotech, Hamburg, Germany) as isotype control. The samples were analyzed using an EPICS XL-MCL flow cytometer (Coulter Immunotech). Based on their forward and side scatter characteristics, living cells were gated. For flow cytometric cell cycle analysis and detection of DNA fragmentation, ∼106 cells were fixed in ice-cold methanol (70%) for 1 h on ice, washed twice with PBS, and resuspended in PBS. Fixed cells were treated with RNase (0.01 mg/ml; Roche Molecular Biochemicals) for 30 min at 37 °C, stained with 50 μg/ml propidium iodine (PI; Sigma), and kept in the dark on ice for 30 min before analysis. Cell cycle analysis was carried out using an EPICS XL-MCL flow cytometer (Coulter Immunotech). Data were analyzed with the Multicycle software (Becton Dickinson, Heidelberg, Germany). Immunohistochemistry—Colonic specimens were immediately frozen and cut into 5-μm sections. HT-29 cells were seeded onto glass tissue slides at different states of confluence. Before staining, slides were fixed in 100% methanol for 15 min and then rinsed twice with PBS. Endogenous peroxidase was quenched for 30 min with 0.3% hydrogen peroxide in PBS buffer. After blocking nonspecific binding sites with normal goat serum (Sigma) for 30 min, the slides were incubated with anti-IL-11Rα mAb, anti-EP-4 mAb (clone Ber-EP-4; DAKO), or anti-mouse IgG1 as negative control for 1 h. Slides were then rinsed with PBS and incubated with biotinylated rabbit anti-mouse IgG (DAKO) for 30 min. After rinsing twice with PBS, slides were treated with streptavidin-conjugated peroxidase (Vector ABC Elite Kit; Vector Laboratories, Burlingame, CA) for 30 min at room temperature. Slides were incubated for 5–10 min in 3,3′-diaminobenzidine solution (Vector Laboratories) at room temperature to allow color development and rinsed with distilled water to quench the reaction. Mayer's hematoxylin was used as a counterstain. Immunoblotting—Cells were resuspended in ice-cold radioimmune precipitation assay buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, 50 mm NaF, and one tablet of complete proteinase inhibitor mixture (Roche Applied Science) per 50 ml) for 10 min, sonicated on ice, and centrifuged (12,000 × g, 15 min at 4 °C). Protein concentration of the supernatant (protein fraction) was determined by Bradford protein assay (Bio-Rad). An aliquot of 35–75 μg of protein was mixed with an equivalent volume of 2× protein loading buffer containing 2-β-mercaptoethanol and boiled for 5 min before loading onto an SDS-polyacrylamide gel. After electrophoresis, proteins were transferred onto nitrocellulose membranes using the XCell Blot Module (Invitrogen BV/NOVEX, Groningen, The Netherlands) and blocked in TBST (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.05% Tween 20) containing 5% nonfat dry milk powder or 3% bovine serum albumin for anti-phosphotyrosine probes. Protein immunoblots were performed using specific antibodies to IL-11Rα, gp130, β-actin (clone JLA20; Calbiochem), phosphotyrosine (Tyr1022/Tyr1023) Jak1 (BIOSOURCE International, Nivelles, Belgium), Jak1 (clone 73, Transduction Laboratories, Lexington, KY), phosphotyrosine (Tyr705) STAT3 (Cell Signaling Technology, Beverly, MA), STAT3 (clone 84; Transduction Laboratories), IκBα (Transduction Laboratories), Bcl-2 (clone 100/D5; BIOSOURCE International), phosphoserine (Ser473) Akt and Akt (both from Cell Signaling Technology). The membranes were further incubated with peroxidase-conjugated secondary antibodies, and protein bands were visualized using a commercial chemiluminescence detection kit (ECL Plus; Amersham Biosciences) as described by the manufacturer. Determination of IL-8 Protein—Supernatants of control and TNF/IL-11-treated cell cultures were collected after 24 h and centrifuged. IL-8 concentration in the incubation medium was quantified by enzyme-linked immunosorbent assay (Biotrak-Amersham, Braunschweig, Germany) according to the manufacturer's protocol. Caspase-9 Fluorometric Assay—CEC in medium with or without IL-11 were kept in suspension by shaking and lysed after the indicated time points. The cell lysate was then tested for protease activity by the addition of the caspase-9-specific substrate peptide LEHD (R&D Systems, Wiesbaden, Germany), which is conjugated to the fluorescent reporter molecule AFC. The cleavage of the peptide by caspase-9 releases the fluorochrom that upon excitation at 400 nm emits light at 505 nm. The level of caspase-9 enzymatic activity in the cell lysate is directly proportional to the fluorescence signal detected with a fluorescence microplate reader. In Vivo Studies in HLA-B27 Transgenic Rats—HLA-B27/β2-microglobulin transgenic rats were obtained from Taconic Inc. (Germantown, WI) and housed in isolated ventilated cages on standard bedding. All rats were fed standard rat chow ad libitum. Recombinant human IL-11 (R&D Systems) was dissolved in sterile PBS containing 0.1% bovine serum albumin to a concentration of 14 μg/ml cytokine. HLA-B27 transgenic rats were divided into two groups (n = 5) and treated either with recombinant human IL-11 or with placebo (0.5 ml subcutaneously, ∼33 μg/kg), in a double blinded fashion, every other day for 2 weeks. The last injection was administered 4 h prior to sacrifice. A TUNEL staining on paraffin-embedded specimen of colonic mucosa was performed as described earlier (46Strater J. Wedding U. Barth T.F. Koretz K. Elsing C. Moller P. Gastroenterology. 1996; 110: 1776-1784Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 47Strater J. Wellisch I. Riedl S. Walczak H. Koretz K. Tandara A. Krammer P.H. Moller P. Gastroenterology. 1997; 113: 160-167Abstract Full Text PDF PubMed Scopus (236) Google Scholar). The number of apoptotic cells was determined microscopically. 1000 epithelial cells were evaluated in three different microscopic fields per animal. Cells were isolated from mucosal specimens as described above (44Rogler G. Daig R. Aschenbrenner E. Vogl D. Schlottmann K. Falk W. Gross V. Scholmerich J. Andus T. Lab. Invest. 1998; 78: 889-890PubMed Google Scholar). Flow cytometrical cell cycle analysis was performed as described for human CEC. Acridine orange staining was performed according to standard procedures. IL-11Rα Is Mainly Expressed on Epithelial Cells within the Human Colon—Since the site of IL-11 action within the intestinal tract had not yet been elucidated, the first aim of our study was to determine the localization of IL-11Rα expression in normal human colonic mucosa with the anti-IL-11Rα E24.2 mAb. Immunohistochemistry clearly revealed IL-11Rα expression as being restricted to epithelial cells (Fig. 1, A and B). No other mucosal cell types were markedly stained by the IL-11Rα mAb. An isotype-matched anti-mouse-IgG1 Ab, used as negative control, did not show any specific staining (Fig. 1, C and D). Epithelial cell characterization on the colonic tissue using the EP-4 mAb demonstrated strong staining of the crypts (Fig. 1, E and F) with the same pattern as was obtained with the anti-IL-11Rα mAb. Since IL-11Rα immunostaining was confined to epithelial cells within the colon mucosa, we investigated receptor expression in the colon cancer cell line HT-29. Cells grown on glass slides at different states of confluence demonstrated a clear staining for the cell surface IL-11 receptor α-chain (Fig. 1G). No detectable staining was obtained when a mouse IgG1 isotype was used as negative control (Fig. 1H). Constitutive IL-11Rα mRNA Expression in HT-29 and CEC—The immunohistochemical findings prompted us to investigate transcription of IL-11Rα mRNA. Total RNA extracte