Characterization of epithelial chemoattractants for human intestinal intraepithelial lymphocytes  T. Shibahara, J.N. Wilcox, T. Couse, J.L. Madara  Gastroenterology  Volume 120, Issue 1, Pages 60-70 (January 2001) DOI: 10.1053/gast.2001.20904 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Stimulation of epithelia by proinflammatory cytokines or PMA alters lymphocyte homing. HT29-19A inverted monolayers were preexposed to various agonists (3–30 ng/mL TNF-α, 10–1000 U/mL IFN-γ, 50–500 U/mL IL-1β, 100–1000 ng/mL PMA, or combination of IFN-γ and TNF-α) for 24 hours. After washing the epithelial monolayers, fluorescently labeled cultured lymphocytes were applied to the basolateral aspect of monolayer for 1 hour (0.3 × 106 lymphocytes/monolayer), and homed lymphocytes were evaluated using the fluorescence plate reader. The data are the mean ± SD. This diagram is representative of 3 separate experiments. *P < 0.05 compared with control migration (unstimulated epithelial monolayer). Gastroenterology 2001 120, 60-70DOI: (10.1053/gast.2001.20904) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Epithelial-conditioned media attract lymphocytes (hatched bar), and this activity is greatly enhanced by preexposure of epithelia to IFN-γ (gray bars) or combination of IFN-γ and TNF-α (stippled bars). Confluent HT29-19A monolayers (5 cm2) were incubated in fresh medium with/without stimulation (3–30 ng/mL TNF-α, 10–1000 U/mL IFN-γ, 50–500 U/mL IL-1β, or combination of IFN-γ and TNF-α) for 24 hours. Conditioned media in this diagram were collected from basolateral side. Insert, the time course of the bioactivity induction by IFN-γ (1000 U/mL). Chemotaxis experiments were performed as described in Materials and Methods. It was estimated that <5% of lymphocytes migrated through the filter without conditioned media (background). Data are the mean ± SD. This diagram is representative of 3 separate experiments. *P < 0.05 compared with chemotaxis by conditioned media from unstimulated epithelia (hatched bar). Gastroenterology 2001 120, 60-70DOI: (10.1053/gast.2001.20904) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Chemotactic activity in epithelial-conditioned media showed protein-like properties, and the response is mediated by a Gαi-coupled receptor(s) on lymphocytes. (A) Checkerboard-like experiments. Epithelial-conditioned media were applied to either the bottom (chemotaxis: migration toward increasing gradients of chemokine) or top (chemokinesis: increased generalized movements of lymphocytes contacting chemokine), and lymphocyte movement was assessed. (B) Conditioned media were subjected to boiling (100°C for 10 minutes) before experimentation. (C) Conditioned media were subjected to precipitation by absolute ethanol, and activity of supernatant and precipitate was assessed. (D) Epithelial monolayer was preexposed to either 2 ng/mL CHX, 1000 U/mL IFN-γ, or combination of 2 ng/mL CHX and 1000 U/mL IFN-γ for 1 day, and chemotactic activities of conditioned media were compared. Alternatively, to examine the direct effect of CHX on lymphocyte migration, 2 ng/mL CHX was added to IFN-γ–stimulated conditioned media just before chemotaxis experiment (shown as IFN-γ [+CHX]). (E) Lymphocytes were preincubated with 2 μg/mL of PTX for 90 minutes at 37°C, and responses to epithelial-conditioned media were assessed. In these experiments, epithelial-conditioned media were from epithelia prestimulated with 1000 U/mL IFN-γ unless otherwise indicated. Data are the mean ± SD. This diagram is representative of 3 separate experiments. *P < 0.05. Gastroenterology 2001 120, 60-70DOI: (10.1053/gast.2001.20904) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Abs to IP-10 or MIG show significant inhibition of IEL chemotaxis toward epithelial conditioned media. HT29-19A basolateral–conditioned media after incubation of 1000 U/mL IFN-γ was used. After background migration was subtracted, each data set was calculated as the ratio of chemotaxis under the presence of each neutralizing Ab against control chemotaxis (no Ab). The data are calculated from 9 to 12 chemotaxis experiments and presented as the mean ± SD. *P < 0.05. Gastroenterology 2001 120, 60-70DOI: (10.1053/gast.2001.20904) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) High level induction of IP-10 and MIG from HT29-19A cells after IFN-γ stimulation. Confluent HT29-19A monolayers (5 cm2) were incubated in fresh medium with/without stimulation (3–30 ng/mL TNF-α, 10–1000 U/mL IFN-γ, 500 U/mL IL-1β, combination of IFN-γ and TNF-α, or 1000 ng/mL of PMA) for 24 hours. Subsequently, concentrations of IP-10, MIG, IL-8, and GRO-α were determined by ELISA. This diagram is representative of 3 separate experiments. (B) Equivalent concentration of recombinant IP-10 or MIG to that found in HT29-19A–conditioned media (determined in Figure 5A) was capable to induce lymphocyte chemotaxis. This diagram is representative of 3 separate experiments. These data are the mean ± SD. *P < 0.05 compared with background migration. Gastroenterology 2001 120, 60-70DOI: (10.1053/gast.2001.20904) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 IP-10 and MIG mRNA are expressed in inflamed intestinal epithelium. mRNA level expression of IP-10 and MIG in vivo was evaluated by in situ hybridization. The photographs are representative of 3 normal colonic tissues and 3 lymphocytic colitis tissues. The tissues were hybridized and developed in parallel and exposed together for 8 weeks. Gastroenterology 2001 120, 60-70DOI: (10.1053/gast.2001.20904) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) Freshly isolated IEL constitituvely express CXCR-3, a common receptor to IP-10 and MIG. CXCR-3 expression on freshly isolated IEL and PBL was examined. Only CD3-positive cells in both populations were analyzed as described in Materials and Methods. The gray histogram shows CXCR-3 expression on fresh IEL or PBL, whereas the open histogram shows the background fluorescence obtained with an irrelevant primary Ab. The histograms are representative of 3 separate experiments. (B) Freshly isolated human IEL show chemotaxis toward epithelial-conditioned media or recombinant IP-10 or MIG. The data are the mean ± SD. The diagram is representative of 3 separate experiments. *P < 0.05 compared with background migration. Gastroenterology 2001 120, 60-70DOI: (10.1053/gast.2001.20904) Copyright © 2001 American Gastroenterological Association Terms and Conditions