Volume 122, Issue 1, Pages 166-177 (January 2002) Fractalkine-mediated signals regulate cell-survival and immune-modulatory responses in intestinal epithelial cells  Stephan Brand, Takanori Sakaguchi, Xiubin Gu, Sean P. Colgan, Hans–Christian Reinecker  Gastroenterology  Volume 122, Issue 1, Pages 166-177 (January 2002) DOI: 10.1053/gast.2002.30329 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 CX3CR1 is expressed in human intestinal epithelial cells. Immunohistochemistry was performed as described in Materials and Methods. (A) CX3CR1-specific antiserum detected the fractalkine receptor in crypt intestinal epithelial cells of the normal human colon (original magnification 40×). (B, C) CX3CR1 was predominantly expressed laterally and apically in intestinal epithelial cells (original magnification 100×). (D) Incubation of colonic frozen sections with preimmune serum (original magnification 100×). (E) Mucosal nerves stained positive for CX3CR1 expression. (F) Corresponding bright field exposure E (original magnification 40×). Gastroenterology 2002 122, 166-177DOI: (10.1053/gast.2002.30329) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Gastrointestinal epithelial tumor–derived cell lines express CX3CR1 and fractalkine. (A) Expression of CX3CR1 and fractalkine mRNA in cell lines as indicated was analyzed by RT-PCR. RT-PCR for CX3CR1, fractalkine, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) yielded PCR products of predicted sizes. (B) Immunoblot analysis of cytosolic and membrane fractions of T84 cells with a CX3CR1-specific antiserum detected a 42-kilodalton band in the membrane fraction (lane 4), which was not detectable in the cytoplasmic fraction (lane 3). This specific band was not found in membrane fractions treated with preimmune serum (lanes 1-2). Gastroenterology 2002 122, 166-177DOI: (10.1053/gast.2002.30329) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Fractalkine activates ERK-MAPKs in T84 cells. Activation and expression of phospho-ERK-1/2, phospho-SAPK/JNK, and phospho-p38 MAPKs were assessed by immunoblotting. (A) Phospho-ERK-1/2 activation after fractalkine stimulation (100 ng/mL). (B) Pretreatment with MEK-1 inhibitor PD98059 (5 μmol/L for 1 hour before fractalkine stimulation) down-regulated phospho-ERK-1/2. (C) Pretreatment with PI3 kinase inhibitor wortmannin (200 nmol/L) did not influence ERK-1/2 activation. (D) Fractalkine stimulation did not activate phospho-SAPK/JNK. P indicates positive control (purchased from New England Biolabs). (E) Fractalkine did not activate phospho-p38. After stripping, all blots were incubated with anti–ERK-1, anti-SAPK/JNK, or anti-p38 antibodies to control for equal loading. One representative experiment of 3 is shown. Gastroenterology 2002 122, 166-177DOI: (10.1053/gast.2002.30329) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Fractalkine-induced ERK-1/2 activation is mediated in part by PTX-sensitive G proteins. Pretreatment of T84 cell with 200 ng/mL PTX for 1 hour decreased fractalkine-induced ERK-MAPK activation by 50%. One representative experiment of 3 is shown. Gastroenterology 2002 122, 166-177DOI: (10.1053/gast.2002.30329) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Fractalkine induces NF-κB consensus sequence binding activity in intestinal epithelial cells. Nuclear extracts of T84 cells were obtained 30 minutes after stimulation with fractalkine (100 ng/mL) or TNF-α (50 ng/mL) and were incubated with antisera or competitors as indicated at the top of the lanes. (A) Nuclear extracts were analyzed by EMSA with an NFκB–specific DNA probe, which revealed nuclear translocation of NF-κB (lane 2), which was inhibited by wild-type competitor (lane 3) but not by a mutant oligonucleotide (lane 4). Lane 1, unstimulated control; lanes 5-7, TNF-α–stimulated controls. (B) NF-κB subunits p50 and p65 are responsible for κB binding: Fractalkine is able to induce the formation of p50 homodimers (complex II) and p65/p50 heterodimers (complex I) in intestinal epithelial cells, and TNF-α stimulation results in p65/p50 heterodimers. (C) Pretreatment with the MEK-1 inhibitor PD98059 (5 μmol/L for 1 hour) down-regulated fractalkine-induced NF-κB activation (lane 2), whereas pretreatment with the PI3 kinase inhibitor wortmannin (200 nmol/L) had little inhibitory effect on fractalkine-induced NF-κB activation (lane 3). Gastroenterology 2002 122, 166-177DOI: (10.1053/gast.2002.30329) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Fractalkine up-regulates fractalkine and IL-8 mRNA expression in intestinal epithelial cells. Northern blot analysis of fractalkine and IL-8 mRNA expression in T84 cells stimulated with fractalkine (100 ng/mL). One representative experiment of 3 is shown. Gastroenterology 2002 122, 166-177DOI: (10.1053/gast.2002.30329) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Fractalkine activates p53 in intestinal epithelial cells. (A) Nuclear extracts of T84 cells were analyzed by EMSA with a p53-specific DNA probe. Extracts were obtained from unstimulated cells (lane 1) and after stimulation with fractalkine (100 ng/mL) for the time intervals indicated (lanes 2-5). Supershift assays show the specificity of this DNA–protein complex, which was supershifted by p53 antibody (lane 6). (B) Competition assays confirmed the specificity of this DNA–protein-binding complex. Nuclear extracts of fractalkine-stimulated T84 cells were incubated with or without competitors (p53 consensus oligonucleotide = wild-type “Wt” or p53 mutant oligonucleotide = mutant “Mut”), as indicated at the top of the lanes. One representative experiment of 3 is shown. (C) Inhibition of fractalkine-induced p53 activation with PD98059, U0126, and PTX. One representative experiment of 2 is shown. Gastroenterology 2002 122, 166-177DOI: (10.1053/gast.2002.30329) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Fractalkine induces intestinal epithelial cell proliferation. T84 cells were grown for 6 days and stimulated on days 2 and 4 with 10 or 100 ng/mL fractalkine or with fractalkine-free medium (negative control). Triplicate samples for each concentration were measured by MTS assay. The cell proliferation rate in the groups treated with fractalkine was 46% and 59%, respectively, higher than in the untreated group. *P = 0.02. Gastroenterology 2002 122, 166-177DOI: (10.1053/gast.2002.30329) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 Fractalkine regulates migration of PMNs into intestinal epithelial cell monolayer. Neutrophil transepithelial migration assays were performed by addition of PMNs to the upper chambers after chemoattractant (100 nmol/L fMLP or recombinant fractalkine in the range of 0.01-10 μg/mL) was added to the opposing (lower) chambers. PMNs (1 × 106) were added, and transmigration was allowed to proceed for 2 hours at 37°C. (A) The chemoattraction of neutrophils to fractalkine corresponded to the expression of CX3CR1 on these cells as determined by flow cytometry. (B) PMNs were able to cross the filter barrier after fractalkine stimulation in the absence of intestinal epithelial cells in a dose-dependent manner. (C) Fractalkine promotes accumulation of PMNs into the T84 cell monolayer in a dose-dependent manner. (D) In contrast to fMLP, fractalkine did not induce transmigration of neutrophils through the intestinal epithelial monolayer. Gastroenterology 2002 122, 166-177DOI: (10.1053/gast.2002.30329) Copyright © 2002 American Gastroenterological Association Terms and Conditions