Volume 121, Issue 2, Pages 246-254 (August 2001) CCR9–Positive lymphocytes and thymus-expressed chemokine distinguish small bowel from colonic Crohn's disease  Konstantinos A. Papadakis, John Prehn, Sofia T. Moreno, Lorna Cheng, Elias A. Kouroumalis, Richard Deem, Tim Breaverman, Paul D. Ponath, David P. Andrew, Peter H.R. Green, Martin R. Hodge, Scott W. Binder, Stephan R. Targan  Gastroenterology  Volume 121, Issue 2, Pages 246-254 (August 2001) DOI: 10.1053/gast.2001.27154 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.1 (A) CCR9 expression on freshly isolated LPMCs from inflamed and adjacent uninvolved small intestinal mucosa from the same donor.Cells were incubated with 3C3 mAb, followed by a cychrome-conjugated goat anti-mouse IgG-(H+ L) secondary antibody and analyzed by FACS.The cells were gated on CD3+ lymphocytes.Representative 1 of 3 independent experiments from different donors with similar results is shown.(B) CCR9 mRNA expression in inflamed intestinal mucosa.Northern blot analysis of CCR9: 32P-labeled CCR9 DNA was used to probe a Northern blot filter (72-hour exposure) from inflamed and uninvolved SB mucosa from the same donor, and inflamed colonic mucosa in UC and Crohn's colitis.Representative 1 of 2 experiments with similar results is shown. Gastroenterology 2001 121, 246-254DOI: (10.1053/gast.2001.27154) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.2 (A) Modulation of CCR9 expression on small bowel lymphocytes upon T-cell receptor activation or IL-2 incubation.Small bowel lymphocytes were incubated with IL-2 (10 U/mL) or PHA (2 μg/mL) for 24 hours, and aliquots of cells were stained with 3C3 (anti-CCR9) mAb, using anti-CD3 and anti-CD4 mAb to directly label the T lymphocytes.Cells were gated on CD3+ lymphocytes.Representative 1 of at least 4 experiments with similar results is shown.(B) CCR9+ T cells are more susceptible to activation-induced cell death.SB LPLs were stained and sorted into CD3+CCR9+ and CD3+CCR9− cells as described in Materials and Methods.The cells were then incubated for 24 hours with PHA (2 μg/mL) and dead (Annexin V+/7AAD+) and apoptotic cells (Annexin V+/7AAD−) were quantitated by flow cytometry.The left lower quadrants (Annexin V−/7AAD−) represent live cells.Representative 1 of 2 experiments with similar results is shown. Gastroenterology 2001 121, 246-254DOI: (10.1053/gast.2001.27154) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.3 (A) CCR9 expression on MLN lymphocytes isolated from draining inflamed and normal SB and colonic mucosa from different donors.Cells were incubated with 3C3 mAb, followed by PE-conjugated anti-IgG2b and analyzed by FACS.The data represent a total of 5 and 7 independent experiments from MLN draining normal and inflamed SB mucosa from different donors, respectively, and 4 experiments each from MLN draining normal and inflamed colon.The bars represent the mean ± SEM.(B) Representative FACS analysis of CCR9 expression on lymphocytes isolated from normal and inflamed SB or colonic MLNs.MLN lymphocytes draining normal and inflamed SB or colon were incubated with 3C3 mAb, and anti-CD3 and anti-CD4 mAb were used to directly label the T lymphocytes. Gastroenterology 2001 121, 246-254DOI: (10.1053/gast.2001.27154) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.4 CCR9 expression on PBLs isolated from patients with SB or colonic CD, normal donors, and patients with active celiac disease.Cells were incubated with 3C3 mAb followed by PE-conjugated anti-IgG2b secondary antibody and analyzed by FACS.The cells were gated on CD3+ lymphocytes.The mean percentage of CD4+CCR9+ PBL in each group is shown. Gastroenterology 2001 121, 246-254DOI: (10.1053/gast.2001.27154) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.5 Expression of TECK protein in inflamed CD intestinal mucosa and thymus (positive control).Fixed sections of inflamed small bowel and colon were stained with anti-TECK mAb (LS202 5A9, IgG1) or isotype control mAb as described in Materials and Methods.(A, C, E, G, I, and J) Staining with anti-TECK mAb.(B, D, F, and H) Staining with an isotype control mAb.(A–H) Small intestine, (I) thymus (positive control), and (J) inflamed colon (CD).(A, arrows) Inflamed SB and (A and C, open arrows) uninvolved SB. Gastroenterology 2001 121, 246-254DOI: (10.1053/gast.2001.27154) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.6 Hypothetical model for the role of TECK and CCR9 in the pathogenesis of small bowel CD.(1) Initiating antigen(s) are sampled by immature dendritic cells of the small intestinal mucosal immune system.(2) After antigen capture and maturation, dendritic cells migrate to draining lymph nodes where they activate T lymphocytes.Activated T cells (3) following expansion are mobilized from the draining LN and, through the thoracic duct, enter the systemic circulation.(4) These CCR9+ T cells are now being detected in the peripheral circulation of patients with small bowel inflammation.(5) These recirculating T cells are preferentially recruited in the SB mucosa possibly through CCR9 interactions with TECK expressed in areas of initial antigen exposure.(6) After their recruitment and in vivo activation, CCR9+ lymphocytes undergo apoptosis and are detected at a lower percentage after isolation and in vitro culture. Gastroenterology 2001 121, 246-254DOI: (10.1053/gast.2001.27154) Copyright © 2001 American Gastroenterological Association Terms and Conditions