Volume 120, Issue 2, Pages 449-459 (February 2001) Disaccharides derived from heparin or heparan sulfate regulate IL-8 and IL-1β secretion by intestinal epithelial cells  Yehuda Chowers, Ofer Lider, Hagai Schor, Iris Barsnack, Ruth Tal, Amiram Ariel, Simon Bar-Meir, Irun R. Cohen, Liora Cahalon  Gastroenterology  Volume 120, Issue 2, Pages 449-459 (February 2001) DOI: 10.1053/gast.2001.21202 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Structure of SSS1-DS composed of glucuronic acid and glucosamine moieties. (B) The heparin-derived DS backbone is composed of an iduronic acid and a glucosamine moiety. The numbers indicate positions to which sulfate or acetyl groups are attached. (C) Summary of the heparin-derived DS used throughout the study. The positions of the sulfate and acetyl molecules are indicated. Note that the heparan sulfate–derived SSS1 and the heparin-derived SSS2 (shown in B) have a similar sulfation pattern, but differ in the DS backbone. Gastroenterology 2001 120, 449-459DOI: (10.1053/gast.2001.21202) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 DS molecules inhibit spontaneous secretion of IL-8 and IL-1β from HT-29 cells. The results are presented as the percent inhibition from baseline IL-8 and IL-1β levels. (A) DS molecules that inhibited IL 8 secretion. The reduction from baseline was significant (P < 0.001) at concentrations ≥ 0.01 ng/mL. (B) DS molecules that did not show dose-dependent inhibition of IL-8 secretion. (C) DS molecules that inhibited IL-1β secretion. The reduction from baseline was significant (P < 0.001) at concentrations ≥ 0.01 ng/mL. (D) DS molecules that did not show a dose-dependent inhibition of IL-1β secretion. Results are the mean and standard deviation (SD) of 3 different experiments. Gastroenterology 2001 120, 449-459DOI: (10.1053/gast.2001.21202) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 DS molecules inhibit spontaneous secretion of IL-8 and IL-1β from Caco-2 cells. The results are presented as the percent inhibition from baseline IL-8 and IL-1β levels. (A) DS molecules that inhibited IL-8 secretion. The reduction from baseline was significant (P < 0.001) at a dose of 0.01 ng/mL for HActH and SSS1, and for the others at ≥0.1 ng/mL. (B) DS molecules that did not inhibit IL-8 secretion. (C) DS molecules that inhibited IL-1β secretion. The reduction from baseline was significant (P < 0.001) at a dose of 0.01 ng/mL for SHH and SSH and for the others at ≥0.1 ng/mL. (D) DS molecules that did not inhibit IL-1β secretion. Results are the mean and SD of 3 different experiments. Gastroenterology 2001 120, 449-459DOI: (10.1053/gast.2001.21202) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 DS molecules inhibit TNF-α–induced (A) IL-8 and (B) IL-1β secretion from HT-29 cells. Cells were grown as confluent monolayers. For experimentation, DS molecules were added for 1 hour, after which TNF-α (200 ng/mL) was added and the cells were incubated for additional 20 hours. The results are presented as the percent inhibition from the cytokine levels that were obtained after TNF-α stimulation. The differences between the effects of SSS2 and SActH were significant at concentrations ≥ 0.1 ng/mL (P < 0.001). Results are the mean and SD of 3 different experiments. Gastroenterology 2001 120, 449-459DOI: (10.1053/gast.2001.21202) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 The inhibitory effect of DS molecules on TNF-α–stimulated cytokine secretion is time dependent. HT-29 cells were grown as confluent monolayers. The cells were treated with DS SSH concurrently, 30 minutes before addition of TNF-α (200 ng/mL), or 30 minutes after TNF-α was added to the culture. The culture was continued for an additional 20 hours, after which the supernatants were collected and assayed for IL-8 and IL-1β levels. (A) IL-8 secretion; (B) IL-1β secretion. The results are presented as the percent inhibition from the cytokine levels that were obtained after TNF-α stimulation. Each result is the mean and SD of 3 different experiments. Gastroenterology 2001 120, 449-459DOI: (10.1053/gast.2001.21202) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 Antagonist DS molecules block the inhibitory effect of DS molecules on IL-8. Antagonist DS molecules were added to the culture medium for 1 hour before the addition of individual inhibitory DS SSH (1 ng/mL). The supernatant was harvested after 24 hours, and IL-8 secretion was assessed using ELISA. The results are presented as the percent inhibition from baseline spontaneous IL-8 levels according to the following equation: % Inhibition = [1 − (Baseline Levels–Levels of DS-Treated Cells)] × 100 (Baseline Levels − Levels of SSH–Treated Cells). (A) Results of experiments performed with HT-29 cells. At 1, 10, and 100 pg/mL, HHH was significantly less antagonistic than were the other molecules (P < 0.0001). (B) Results of experiments performed with Caco-2 cells. Results represent the mean and SD of 3 different experiments. Gastroenterology 2001 120, 449-459DOI: (10.1053/gast.2001.21202) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 Treatment with DS molecules does not down-regulate IL-8 mRNA levels. HT-29 cells were cultured to confluence. DS molecules were added to the culture for 1 hour, after which TNF-α was added for an additional 2 hours. After culture, the cells were harvested and the RNA was extracted and analyzed by an RNA protection assay. The appropriate bands corresponding to IL-8 and GAPDH were measured by a PhosphorImager; the ratio between them is presented. Gastroenterology 2001 120, 449-459DOI: (10.1053/gast.2001.21202) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 8 Treatment with DS molecules suppresses secretion of IL-8 and IL-1β from HT-29 cells, but their levels remain elevated within the cells HT-29 cells were grown to confluence. The cells were treated with DS molecules or somatostatin (10−6 mol/L) for 1 hour, after which TNF-α (200 ng/mL) was added for 24 hours. After culture, the supernatants were collected and the cells were lysed by 3 freeze and thaw cycles. Both lysate and supernatants were assayed by ELISA for (A) IL-8 and (B) IL-1β levels. The results are shown as the mean and SD of 3 different experiments and are presented as the percent inhibition of IL-8 or IL-1β levels relative to their basal levels after treatment with TNF-α. Gastroenterology 2001 120, 449-459DOI: (10.1053/gast.2001.21202) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 9 Treatment with DS inhibits membrane translocation of IL-8. HT-29 cells were grown to confluence, then treated for 1 hour with an inhibitory DS (SSS2) or a noninhibitory DS (SActH), after which TNF-α was added for an overnight incubation. After culture, the cells were stained for IL-8 expression. (A) TNF-α–stimulated cells; (B) cells treated with a noninhibitory DS; (C) cells treated with an inhibitory DS; (D) cells stained with second antibody only. A representative experiment of 2. Gastroenterology 2001 120, 449-459DOI: (10.1053/gast.2001.21202) Copyright © 2001 American Gastroenterological Association Terms and Conditions