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• W2040754829 abstract "In inflammatory conditions of the gut, cytokines are released into the mucosa and submucosa propagating and sustaining the inflammatory response. In CaCo-2 cells, we have shown that various inflammatory cytokines interfere with the secretion of lipids, an effect that is likely caused by the release of a ligand to the epidermal growth factor (EGF) receptor. In the present study, the role of the EGF receptor signaling pathway and the effects of the cytokines tumor necrosis factor-α (TNF-α) and and interleukin 1β (IL-1β) on triacylglycerol-rich lipoprotein secretion were investigated. CaCo-2 cells were incubated with oleic acid to enhance triacylglycerol-rich lipoprotein secretion. TNF-α and IL-1β significantly decreased the basolateral secretion of apolipoprotein B (apoB) mass, with IL-1β being more potent. Tyrphostin, an inhibitor of the EGF receptor intrinsic tryosine kinase, prevented or markedly attenuated the decrease in apoB secretion by TNF-α or IL-1β. Both cytokines increased the phosphorylation of the EGF receptor by 30 min. Moreover, phosphotyrosine immunoblots of the EGF receptor demonstrated an increase in tyrosine residues phosphorylated by 0.5 and 6.5 h. At both these time points, TNF-α and IL-1β also decreased the binding of EGF to its cell surface receptor. At 6.5 h, activation of the EGF receptor was sustained. In contrast, the early activation of the receptor was only transient as receptor phosphorylation and binding of EGF to its receptor returned to basal levels by 2 h. Preventing ligand binding to the EGF receptor by a receptor-blocking antibody attenuated receptor activation observed after 6.5 h. This did not occur at 0.5 h, suggesting that early activation of the EGF receptor was non-ligand-mediated. Similarly, apoB secretion was inhibited by an early non-ligand-mediated process; whereas at the later time, inhibition of apoB secretion was ligand-mediated. Thus, the inflammatory cytokines TNF-α and IL-1β interfere with the secretion of triacylglycerol-rich lipoproteins by both early and delayed signaling events mediated by the EGF receptor signaling pathway. In inflammatory conditions of the gut, cytokines are released into the mucosa and submucosa propagating and sustaining the inflammatory response. In CaCo-2 cells, we have shown that various inflammatory cytokines interfere with the secretion of lipids, an effect that is likely caused by the release of a ligand to the epidermal growth factor (EGF) receptor. In the present study, the role of the EGF receptor signaling pathway and the effects of the cytokines tumor necrosis factor-α (TNF-α) and and interleukin 1β (IL-1β) on triacylglycerol-rich lipoprotein secretion were investigated. CaCo-2 cells were incubated with oleic acid to enhance triacylglycerol-rich lipoprotein secretion. TNF-α and IL-1β significantly decreased the basolateral secretion of apolipoprotein B (apoB) mass, with IL-1β being more potent. Tyrphostin, an inhibitor of the EGF receptor intrinsic tryosine kinase, prevented or markedly attenuated the decrease in apoB secretion by TNF-α or IL-1β. Both cytokines increased the phosphorylation of the EGF receptor by 30 min. Moreover, phosphotyrosine immunoblots of the EGF receptor demonstrated an increase in tyrosine residues phosphorylated by 0.5 and 6.5 h. At both these time points, TNF-α and IL-1β also decreased the binding of EGF to its cell surface receptor. At 6.5 h, activation of the EGF receptor was sustained. In contrast, the early activation of the receptor was only transient as receptor phosphorylation and binding of EGF to its receptor returned to basal levels by 2 h. Preventing ligand binding to the EGF receptor by a receptor-blocking antibody attenuated receptor activation observed after 6.5 h. This did not occur at 0.5 h, suggesting that early activation of the EGF receptor was non-ligand-mediated. Similarly, apoB secretion was inhibited by an early non-ligand-mediated process; whereas at the later time, inhibition of apoB secretion was ligand-mediated. Thus, the inflammatory cytokines TNF-α and IL-1β interfere with the secretion of triacylglycerol-rich lipoproteins by both early and delayed signaling events mediated by the EGF receptor signaling pathway. epidermal growth factor transforming growth factor tumor necrosis factor interleukin monoclonal antibody bovine serum albumin polyacrylamide gel electrophoresis N-acetylneuraminylgalactosylceramide Inflammatory conditions of the small intestine, such as gluten-sensitive enteropathy or Crohn's disease, can result in mucosal damage leading to malabsorption of nutrients (1.Kagnoff M.F. Gastroenterol. Clin. North Am. 1992; 21: 405-425Abstract Full Text PDF PubMed Google Scholar). The localized release of inflammatory cytokines into the mucosa and submucosa likely mediates and perpetuates the inflammatory response (2.Fiocchi C. Immunol. Res. 1991; 10: 239-246Crossref PubMed Scopus (20) Google Scholar). Lymphocytes, monocyte/macrophages, and mast cells that infiltrate the mucosa are the major source of these inflammatory peptides (3.Brynskov J. Nielsen O.H. Ahnfelt-Ronne I. Bendtzen K. Scand. J. Gastroenterol. 1992; 27: 897-906Crossref PubMed Scopus (63) Google Scholar, 4.Braegger C.P. MacDonald T.T. Ann. Allergy. 1994; 72: 135-141PubMed Google Scholar). Recent evidence, however, demonstrates that intestinal epithelial cells also synthesize and secrete a number of inflammatory cytokines (5.Eckmann L. Jung H.C. Schurer-Maly C. Panja A. Morzycka-Wroblewska E. Kagnoff M.F. Gastroenterology. 1993; 105: 1689-1697Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 6.Radema S.A. van Deventer S.J. Cerami A. Gastroenterology. 1991; 100: 1180-1186Abstract Full Text PDF PubMed Google Scholar, 7.McGee D.W. Elson C.O. McGhee J.R. Infect. Immun. 1993; 61: 4637-4644Crossref PubMed Google Scholar, 8.Shirota K. LeDuy L. Yuan S.Y. Jothy S. Virchows Arch. B. Cell Pathol. Incl. Mol. Pathol. 1990; 58: 303-308Crossref PubMed Scopus (138) Google Scholar, 9.Beil W.J. Weller P.F. Peppercorn M.A. Galli S.J. Dvorak A.M. J. Leukocyte Biol. 1995; 58: 284-298Crossref PubMed Scopus (98) Google Scholar, 10.Przemioslo R.T. Kontakou M. Nobili V. Ciclitira P.J. Gut. 1994; 35: 1398-1403Crossref PubMed Scopus (95) Google Scholar). Moreover, because they possess receptors for several cytokines (8.Shirota K. LeDuy L. Yuan S.Y. Jothy S. Virchows Arch. B. Cell Pathol. Incl. Mol. Pathol. 1990; 58: 303-308Crossref PubMed Scopus (138) Google Scholar, 11.Schuerer-Maly C.C. Eckmann L. Kagnoff M.F. Falco M.T. Maly F.E. Immunology. 1994; 81: 85-91PubMed Google Scholar, 12.Andoh A. Fujiyama Y. Bamba T. Hosoda S. J. Immunol. 1993; 151: 4239-4247PubMed Google Scholar, 13.Molmenti E.P. Ziambaras T. Perlmutter D.H. J. Biol. Chem. 1993; 268: 14116-14124Abstract Full Text PDF PubMed Google Scholar), it is likely that enterocytes, by interacting directly with inflammatory cytokines, participate in and contribute to the various pathophysiological derangements observed in inflammatory conditions of the gut. Evidence in support of such a notion was demonstrated by the ability of inflammatory cytokines to up-regulate the synthesis of acute phase proteins (13.Molmenti E.P. Ziambaras T. Perlmutter D.H. J. Biol. Chem. 1993; 268: 14116-14124Abstract Full Text PDF PubMed Google Scholar) and complement factors (12.Andoh A. Fujiyama Y. Bamba T. Hosoda S. J. Immunol. 1993; 151: 4239-4247PubMed Google Scholar) in cultured human intestinal cells. Cytokines, however, are not the only mediators of inflammation which are released into the mucosa under conditions of inflammation. Several other bioactive molecules such as growth factors, prostaglandins, and reactive oxygen species are secreted by intestinal epithelial cells (14.Sartor R.B. Gastroenterology. 1994; 106: 533-539Abstract Full Text PDF PubMed Google Scholar). Together with cytokines, these factors act coordinately to regulate the extent of mucosal injury as well as mediate tissue repair. Growth factors EGF1 and TGF-α, ligands to the transmembrane EGF receptor present on intestinal epithelial cells, have been shown to play a significant role in the restitution of mucosal damage in the gut (15.Tarnawski A.S. Jones M.K. J. Clin. Gastroenterol. 1998; 27: S12-S20Crossref PubMed Scopus (94) Google Scholar, 16.Schultz G. Rotatori D.S. Clark W. J. Cell. Biochem. 1991; 45: 346-352Crossref PubMed Scopus (295) Google Scholar). Ligand-mediated activation of the EGF receptor triggers a cascade of events resulting in enhanced cell migration and proliferation that serve to repair the denuded surface of the mucosa. Moreover, by mediating the increased production of mucopolysaccharides, prostaglandins, and extracellular matrix components, the EGF receptor likely plays an important role in protecting mucosal surfaces from further injury (17.Konturek J.W. Brzozowski T. Konturek S.J. J. Clin. Gastroenterol. 1991; 13: S88-S97Crossref PubMed Scopus (67) Google Scholar). By modulating transport processes such as ion exchange (18.Donowitz M. Montgomery J.L. Walker M.S. Cohen M.E. Am. J. Physiol. 1994; 266: G647-G656PubMed Google Scholar, 19.Opleta-Madsen K. Hardin J. Gall D.G. Am. J. Physiol. 1991; 260: G807-G814PubMed Google Scholar, 20.Horvath K. Hill I.D. Devarajan P. Mehta D. Thomas S.C. Lu R.B. Lebenthal E. Biochim. Biophys. Acta. 1994; 1222: 215-222Crossref PubMed Scopus (37) Google Scholar) and glucose absorption (19.Opleta-Madsen K. Hardin J. Gall D.G. Am. J. Physiol. 1991; 260: G807-G814PubMed Google Scholar, 20.Horvath K. Hill I.D. Devarajan P. Mehta D. Thomas S.C. Lu R.B. Lebenthal E. Biochim. Biophys. Acta. 1994; 1222: 215-222Crossref PubMed Scopus (37) Google Scholar, 21.Bird A.R. Croom Jr., W.J. Fan Y.K. Daniel L.R. Black B.L. McBride B.W. Eisen E.J. Bull L.S. Taylor I.L. J. Nutr. 1994; 124: 231-240Crossref PubMed Scopus (43) Google Scholar) the receptor might also play a significant role in regulating intestinal cell function during inflammation. The role of the EGF receptor in decreasing the absorptive function of enterocytes in inflammation, however, has not been investigated. Cytokines that are considered proinflammatory, under certain conditions, may also suppress inflammation and promote wound healing and repair (22.Sartor R.B. Immunol. Res. 1991; 10: 465-471Crossref PubMed Scopus (64) Google Scholar, 23.Wahl S.M. McCartney-Francis N. Mergenhagen S.E. Immunol. Today. 1989; 10: 258-261Abstract Full Text PDF PubMed Scopus (408) Google Scholar). Similar to the EGF receptor, they have been shown to mediate cellular functions such as proliferation, differentiation, deposition of extracellular matrix, and cell motility. Thus, together with the EGF receptor signaling pathway, cytokines likely modulate intestinal epithelial cell function during inflammation. In a previous study, we demonstrated that certain inflammatory cytokines interfered with normal intestinal lipoprotein synthesis and secretion (24.Murthy S. Mathur S.N. Varilek G. Bishop W. Field F.J. Am. J. Physiol. 1996; 270: G94-G102Crossref PubMed Google Scholar). In the present study, we addressed whether cytokines inhibit the secretion of lipoproteins by activating the EGF receptor signaling pathway. The effects of two inflammatory cytokines, TNF-α and IL-1β, on EGF receptor activation and triacylglycerol-rich lipoprotein secretion were studied in a cultured human intestinal cell line, CaCo-2. The results demonstrate that TNF-α and IL-1β inhibit the secretion of triacylglycerol-rich lipoproteins by both a rapid and delayed activation of the EGF receptor. This occurs by non-ligand- and ligand-mediated mechanisms, respectively. Recombinant human TNF-α and IL-1β were purchased from R & D Systems (Minneapolis). Carrier-free EGF was purchased from Becton Dickinson (Bedford, MA). Horseradish peroxidase substrate, SuperSignal West Femto maximum sensitivity substrate kit, and IODO-GEN were purchased from Pierce. Rabbit polyclonal antibody to human EGF receptor was from Upstate Biotechnology Inc. (Lake Placid, NY). Mouse monoclonal anti-phosphotyrosine antibody, mouse monoclonal anti-EGF receptor-blocking antibody (mAb 528), goat anti-mouse IgG conjugated to horseradish peroxidase, and protein A+G-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-human apoB polyclonal antibody and tyrphostin AG1478 were obtained from Calbiochem. Mouse monoclonal antibody to human apoB and rabbit anti-human apoB polyclonal antibody conjugated to horseradish peroxidase were bought from Biodesign (Kennebunkeport, ME). Recombinant protein A-Sepharose was purchased from Repligen (Cambridge, MA). Oleic acid, BSA, and Glycerol Phosphate Oxidase Trinder Kit were purchased from Sigma. A TMB microwell peroxidase substrate system containing 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide was purchased from Kirkegaard and Perry (Gaithersburg, MD). Nunc 96-well immunoplates were obtained from PGC Scientific (Gaithersburg, MD). CellTiter 96 was from Promega (Madison, WI). 32Pi (6,000 Ci/mmol) was purchased from NEN Life Science Products. Carrier-free 125I (100 mCi/ml) was purchased from ICN Biomedicals Inc. (Costa Mesa, CA). CaCo-2 cells were cultured on T-75 flasks (Corning Glassworks, Corning, NY) in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 4.5 g/liter glucose and supplemented with 10% fetal bovine serum (Summit Biotechnology, Fort Collins, CO), 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 50 μg/ml gentamicin. Once the flasks reached 80% confluence, the cells were split and seeded at a density of 0.2 × 105 cells/well onto polycarbonate micropore membranes (0.4-μm pore size, 6.5-mm diameter) inserted into transwells (Costar, Cambridge, MA). For experiments in which triacylglycerol mass, EGF receptor phosphorylation, and cell surface125I-EGF binding were estimated, cells were subcultured in 24-mm diameter transwells. Cells were fed every other day and were used 14 days after seeding. On the day of the experiment, cells were washed with media, and cytokines were added to the lower chambers in serum-free Hanks' balanced saline solution and 1 m HEPES (HBSS) or M199 and 1m HEPES (M199) containing 0.1% BSA. Control cells received medium containing 0.1% BSA alone. All cells received 250 μm oleic acid and 62.5 μm BSA in the apical chamber. Incubations were carried out for 18 h or less at 37 °C in an atmosphere of 95% compressed air and 5% CO2. Cell viability and proliferation were assessed by measuring the activity of mitochondrial dehydrogenase using the CellTiter 96 assay kit as described previously (24.Murthy S. Mathur S.N. Varilek G. Bishop W. Field F.J. Am. J. Physiol. 1996; 270: G94-G102Crossref PubMed Google Scholar). This assay is based on the mitochondrial conversion of a tetrazolium salt into a blue formazan product that is released into the medium. After an overnight incubation with the treatments, the release of the colored formazan dye into the medium was measured spectrophotometrically. Compared with control cells, the relative absorbance of the dye released from cells incubated with TNF-α or IL-1β was 0.97 ± 0.09 or 0.96 ± 0.13, respectively. ApoB mass in cells and basal media was determined by sandwich enzyme-linked immunosorbent assay as described previously (24.Murthy S. Mathur S.N. Varilek G. Bishop W. Field F.J. Am. J. Physiol. 1996; 270: G94-G102Crossref PubMed Google Scholar). The presence of the treatments in the media did not interfere with the estimation of apoB mass by the enzyme-linked immunosorbent assay. Cells were incubated for 18 h with 500 μCi of 32Pi/well in phosphate-free Dulbecco's modified Eagle's medium. Treatments were added to the basal wells in the continued presence of labeled inorganic phosphate. After incubation, cells were rinsed in ice-cold phosphate-buffered saline and scraped and lysed in 1 ml of radioimmune precipitation buffer containing 1 mm phenylmethanesulfonyl fluoride, 21 μm leupeptin, 2 mm benzamidine, 30 μl/ml aprotinin, 1 mg/ml soybean trypsin inhibitor, 2 mm sodium orthovanadate, 20 μm sodium pyrophosphate, and 20 μm sodium fluoride. The cell lysates were precleared by shaking for 1 h at 4 °C with protein A-Sepharose followed by a quick high speed centrifugation. EGF receptor was immunoprecipitated from the precleared supernatants by incubating for 18 h with 1 μg/ml rabbit anti-human EGF receptor antibody. The antigen-antibody complexes were precipitated by incubating with protein A+G-agarose for 1 h at room temperature followed by a brief high speed centrifugation. The immunoprecipitates were washed extensively with phosphate-buffered saline and the EGF receptor protein dissociated from the antibody-antigen complex with 30 μl of 2 × Laemmli sample buffer and 15 μl of 0.2 m glycine buffer (pH 2). The protein was resolved by SDS-PAGE on 8% porous gels as described previously (25.Doucet J.P. Murphy B.J. Tuana B.S. Anal. Biochem. 1990; 190: 209-211Crossref PubMed Scopus (56) Google Scholar). Gels were fixed with 7% acetic acid and 5% methanol, dried, and exposed to x-ray film for 8 h. The incorporation of labeled inorganic phosphate into the EGF receptor was estimated by scanning the gels on Ambis 4000 biological image analyzer (Scanalytics, Billerica, MA). EGF was iodinated using IODO-GEN reagent as prescribed by Pierce. After treatment with TNF-α or IL-1β, binding of 125I-EGF to cell surface EGF receptors was estimated as described previously (26.Bishop W.P. Wen J.T. Am. J. Physiol. 1994; 267: G892-G900PubMed Google Scholar). Cells were washed with M199 and incubated for 2 h at 4 °C with 0–1000 ng/ml iodinated EGF (0.0003 μCi/ng). From previous experiments it was found that the binding of 100 ng/ml radiolabeled EGF to cell surface EGF receptors plateaus after 2 h of incubation. EGF was diluted in M199 containing 0.1% BSA and added to the lower wells. M199 was added to the upper chambers. After extensive washing with ice-cold M199 containing 0.1% BSA followed by several rinses with M199 alone, cells were scraped in 1 ml of radioimmune precipitation buffer and counted in a gamma counter. Nonspecific binding was estimated by incubating cells with 5 μg/ml cold EGF in the presence of 50 ng/ml 125I-EGF and did not exceed 5% of the total binding of labeled EGF. The specificity of the binding of EGF to its cell surface receptor in CaCo-2 cells was determined by Bishop and Wen (26.Bishop W.P. Wen J.T. Am. J. Physiol. 1994; 267: G892-G900PubMed Google Scholar), who demonstrated that the binding of iodinated EGF to cell surfaces is abolished in the presence of the EGF receptor-blocking antibody, mAb 528. Furthermore, when cells were incubated at 4 °C with labeled EGF and then exposed to bis(sulfosuccinimidyl)suberate (Pierce) to cross-link the ligand to its receptor, more than 80% of the EGF bound to cell surfaces was recovered in a band corresponding to the EGF receptor (data not shown). In experiments in which cells were incubated with the cytokines in the presence of mAb 528, the blocking antibody bound to cell surfaces was removed prior to estimation of cell surface binding of labeled EGF. This was accomplished by extensively washing the cells with 100 mm sodium chloride and 500 mm glycine, pH 3, followed by several rinses with M199. Subsequent binding of labeled EGF to control cells incubated with or without mAb 528 was similar, indicating therefore that the stringent wash protocol effectively removed the bound monoclonal antibody from cell surfaces. After incubation with the treatments, EGF receptor was immunoprecipitated from precleared cell lysates as described above. The receptor protein was dissociated from the antigen-antibody complex, separated by SDS-PAGE, and electroblotted onto polyvinylidene difluoride membranes at 15 V for 18 h. The membranes were blocked for 1 h at 37 °C in phosphate-buffered saline (10 mm sodium phosphate, 100 mm sodium chloride, pH 7.4) containing 5% non-fat dry milk, 5% normal goat serum, and 0.1% Tween 20 (blocking buffer). The membranes were then incubated for 1 h with mouse monoclonal anti-phosphotyrosine antibody diluted 20,000-fold in the blocking buffer. After washing with phosphate-buffered saline containing 0.1% Tween 20, the membranes were incubated for 1 h at room temperature with goat anti-mouse IgG-horseradish peroxidase diluted 50,000-fold in blocking buffer. After extensive washing, the membranes were incubated with horseradish peroxidase chemiluminescent substrates, wrapped in Saran Wrap, and then exposed to x-ray film. Band densities were scanned on Hewlett-Packard ScanJet IIcx/T scanner, Hewlett Packard (Greely, CO) and quantitated with the computer-assisted program, Sigma Gel, Jandel Scientific (San Rafael, CA). Total protein content in cells was determined by the method of Lowry et al. (27.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Triacylglycerol mass in cells was measured using the Glycerol Phosphate Oxidase Trinder Kit as described previously (28.Murthy S. Albright E. Mathur S.N. Field F.J. Biochim. Biophys. Acta. 1990; 1045: 147-155Crossref PubMed Scopus (43) Google Scholar). Statistical analyses of data were performed by analysis of variance, Tukey's t test, Student's t test, the least squares method of determining the best fitting straight line, and small sample t tests for parallelism and common intercepts (29.Kleinbaum D.G. Kupper L.L. Applied Regression Analysis and Other Multivariate Methods. Duxbury Press, Wadsworth Publishing Company, Belmont, CA1978: 15-112Google Scholar). In a previous study, we demonstrated that TNF-α and IL-1β decreased the basolateral secretion of apoB by CaCo-2 cells in the absence of fatty acids, experimental conditions that do not promote the secretion of triacylglycerol-rich lipoproteins (24.Murthy S. Mathur S.N. Varilek G. Bishop W. Field F.J. Am. J. Physiol. 1996; 270: G94-G102Crossref PubMed Google Scholar). To address the regulation of triacylglycerol-rich lipoprotein secretion by TNF-α and IL-1β, CaCo-2 cells were incubated for 18 h with increasing concentrations of TNF-α or IL-1β and 250 μm oleic acid. Oleic acid, at this concentration, has been demonstrated in CaCo-2 cells to stimulate the secretion of lipoproteins enriched in triacylglycerols (30.Field F.J. Albright E. Mathur S.N. J. Lipid Res. 1988; 29: 1427-1437Abstract Full Text PDF PubMed Google Scholar). After the incubation, the mass of apoB within cells and that secreted into the basolateral medium was estimated. Both TNF-α and IL-1β decreased the secretion of apoB with IL-1β being the more potent cytokine (Table I). Compared with control cells, IL-1β, at concentrations of 0.001 and 0.1 ng/ml, decreased apoB secretion by 30 and 60%, respectively. Higher concentrations of IL-1β did not decrease apoB secretion further. In contrast, compared with the effects of IL-1β, TNF-α decreased apoB secretion to a similar degree but required much higher concentrations, 10 and 100 ng/ml, respectively. Neither TNF-α nor IL-1β altered the amount of apoB mass within cells.Table IEffect of TNF-α and IL-1β on the basolateral secretion of apoB in CaCo-2 cellsBasal mediaCellsapoB ng/wellControl221 ± 420 ± 1TNF-α (ng/ml) 5184 ± 5*19 ± 4 10147 ± 6*22 ± 3 50118 ± 8*21 ± 2 100107 ± 5*25 ± 3IL-1β (ng/ml) 0.001158 ± 5*17 ± 1 0.01138 ± 3*16 ± 2 0.191 ± 7*16 ± 1 193 ± 7*16 ± 3 1098 ± 8*18 ± 2 10095 ± 8*17 ± 2CaCo-2 cells were incubated for 18 h with increasing concentrations of TNF-α or IL-1β dissolved in HBSS containing 0.1% BSA. Control cells received HBSS plus BSA alone. Cytokines were added to the lower well. The upper well contained 250 μm oleic acid complexed to 62.5 μm BSA. The accumulation of apoB in the lower wells and in cells was estimated by sandwich enzyme-linked immunosorbent assay as described under “Experimental Procedures.” Results are expressed as mean ± S.E. of six wells/treatment.* p < 0.01 compared with control cells. Open table in a new tab CaCo-2 cells were incubated for 18 h with increasing concentrations of TNF-α or IL-1β dissolved in HBSS containing 0.1% BSA. Control cells received HBSS plus BSA alone. Cytokines were added to the lower well. The upper well contained 250 μm oleic acid complexed to 62.5 μm BSA. The accumulation of apoB in the lower wells and in cells was estimated by sandwich enzyme-linked immunosorbent assay as described under “Experimental Procedures.” Results are expressed as mean ± S.E. of six wells/treatment. * p < 0.01 compared with control cells. A decrease in apoB secretion by cells incubated with either cytokine suggested that TNF-α and IL-1β caused a decrease in the number of lipoprotein particles being secreted. To address the effect of the cytokines on the amount of triacylglycerols carried per lipoprotein particle, cells were incubated for 18 h with oleic acid and 100 ng/ml TNF-α or 10 ng/ml IL-1β. The amount of triacylglycerols within cells and that secreted into the basolateral medium was then estimated. Compared with control cells, the secretion of triacylglycerols by cells incubated with TNF-α was decreased modestly (23.61 ± 2.39 versus 19.48 ± 1.09 μg/mg protein, n = 6, p < 0.05). In cells incubated with IL-1β, less triacylglycerol was secreted compared with control cells or cells incubated with TNF-α (23.61 ± 2.39versus 16.04 ± 0.03 μg/mg protein, p< 0.01, versus TNF-α p < 0.05). Neither TNF-α nor IL-1β altered the amount of triacylglycerols within cells (control cells, 58.71 ± 1.7; TNF-α cells, 61.53 ± 2.37; IL-1β cells, 62.48 ± 2.98 μg/mg; n = 6). Thus TNF-α and IL-1β decrease the secretion of triacylglycerol-rich lipoproteins. We have demonstrated previously that ligand-mediated activation of the EGF receptor decreases the secretion of lipids and apoB in CaCo-2 cells (31.Murthy S. Mathur S. Bishop W.P. Field E.J. J. Lipid Res. 1997; 38: 206-216Abstract Full Text PDF PubMed Google Scholar). Because TNF-α and IL-1β have been observed to modulate the function of the EGF receptor in human fibroblasts (32.Bird T.A. Saklatvala J. J. Immunol. 1989; 142: 126-133PubMed Google Scholar), we addressed the possibility that TNF-α and IL-1β interfered with triacylglycerol-rich lipoprotein secretion in CaCo-2 cells by activating the EGF receptor. Intrinsic tyrosine kinase activity of the EGF receptor is essential for EGF receptor-mediated signaling events (33.Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4594) Google Scholar). To investigate the role of the EGF receptor signaling pathway in apoB secretion, EGF receptor tyrosine kinase activity was inhibited by tyrphostin AG1478 (34.Levitzki A. Gazit A. Science. 1995; 267: 1782-1788Crossref PubMed Scopus (1616) Google Scholar). CaCo-2 cells were incubated for 18 h with oleic acid and increasing concentrations of TNF-α or IL-1β in the presence or absence of tyrphostin AG1478. The amount of apoB secreted was then estimated. As shown in Fig.1 A, tyrphostin completely prevented the decrease in apoB secretion observed in cells incubated with TNF-α alone. In cells incubated with IL-1β, tyrphostin significantly attenuated the decrease in apoB secretion. Moreover, tyrphostin completely blocked the decrease in apoB secretion observed in cells incubated with EGF, a ligand of the EGF receptor which activates receptor tyrosine kinase. Tyrphostin itself had no effect on the secretion of apoB. These results suggest that the cytokines decreased apoB secretion by activating the EGF receptor signaling pathway. This was addressed further by incubating cells for 18 h with 100 ng/ml TNF-α, 10 ng/ml IL-1β, or both. In addition, some cells were also incubated with 100 ng/ml EGF alone or together with either TNF-α or IL-1β. This concentration of EGF causes saturation of cell surface binding (see Fig. 3 A) and likely maximal stimulation of the EGF receptor signaling pathway. At the end of the incubation, the amount of apoB secreted was estimated. The results are shown in Fig. 1 B. ApoB secretion by cells incubated with EGF was decreased dramatically and was 2-fold less than the amount secreted by cells incubated with either cytokine alone. When cells were incubated with both cytokines together, the decrease in apoB secretion was additive and was similar to the inhibition induced by EGF alone. It is likely, therefore, that together TNF-α and IL-1β act additively to activate the EGF receptor signaling pathway leading to an inhibition in apoB secretion. Moreover, compared with the effects of EGF alone, addition of either TNF-α or IL-1β to cells incubated with EGF did not decrease the secretion of apoB further, suggesting that the cytokines were acting through the same pathway as EGF in inhibiting apoB secretion.Figure 3Effect of TNF -α or IL-1β on cell surface binding of EGF.Panel A, cells were incubated for 30 min with either 100 ng/ml TNF-α (▪) or 10 ng/ml IL-1β (▴). The cytokines were added to the lower wells in HBSS containing 0.1% BSA. Control cells (○) received medium containing BSA alone. 250 μm oleic acid complexed to 62.5 μm BSA was added to the upper wells. At the end of the incubation, cells were washed thoroughly with ice-cold M199, and the basolateral medium was replaced with M199 containing 0.1% BSA and increasing concentrations of 125I-EGF (0.1–1000 ng/ml, 0.0003 μCi/ng EGF). M199 was added to the upper wells. After 2 h of incubation at 4 °C, cells were washed thoroughly to remove unbound labeled EGF, lysed, and counted by gamma counting to estimate cell-associated labeled EGF. Nonspecific binding of labeled EGF to cell surfaces was estimated by incubating cells with 5 μg/ml cold EGF in the presence of 50 ng/ml labeled EGF. Results are expressed as the mean ± S.E. of bound EGF detected/mg of protein.Panel B, Scatchard plot analysis of the results presented inpanel A. Symbols are the same as in panel A. Panel C, cells were incubated for increasing time" @default.
• W2040754829 created "2016-06-24" @default.
• W2040754829 creator A5049488339 @default.
• W2040754829 creator A5089106819 @default.
• W2040754829 creator A5090039729 @default.
• W2040754829 date "2000-03-01" @default.
• W2040754829 modified "2023-10-13" @default.
• W2040754829 title "Tumor Necrosis Factor-α and Interleukin-1β Inhibit Apolipoprotein B Secretion in CaCo-2 Cells via the Epidermal Growth Factor Receptor Signaling Pathway" @default.
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