Volume 122, Issue 3, Pages 734-744 (March 2002) Intravital observation of adhesion of lamina propria lymphocytes to microvessels of small intestine in mice  Hitoshi Fujimori, Soichiro Miura, Seiichiro Koseki, Ryota Hokari, Shunsuke Komoto, Yuriko Hara, Satoshi Hachimura, Shuichi Kaminogawa, Hiromasa Ishii  Gastroenterology  Volume 122, Issue 3, Pages 734-744 (March 2002) DOI: 10.1053/gast.2002.31899 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Expression of adhesion molecules (L-selectin, α4-integrin, β7-integrin, CD11a, and αE-integrin), CD69, and CD45RB in lamina propria (LPLs) and splenic (SPLs) T lymphocytes determined by flow cytometric analysis. Lymphocytes (2 × 105) were first incubated with anti-mouse monoclonal antibodies against L-selectin (MEL-14), α4-integrin (R1-2), LPAM-1 (DATK-32) β7-integrin (FIB27), CD11a (M17/4), CD69 (H1-2F3), CD45RB (16A), and αE-integrin (M290). They were then incubated with 1 mL of FITC-labeled anti-rat IgG and anti-hamster IgG. Flow cytometric analysis was performed using FACSort (Becton Dickinson, Mountain View, CA). Data on viable cells, as determined by forward light scatter intensity, were obtained using CONSORT software. Representative data from at least 4 individual measurements are shown. Gastroenterology 2002 122, 734-744DOI: (10.1053/gast.2002.31899) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Representative photomicrographs showing the distribution of CFSE-labeled lamina propria T lymphocytes (LPLs) (left) and splenic T lymphocytes (SPLs) (right) in the postcapillary venules of Peyer's patches 30 minutes after infusion. SPLs and LPLs (1 × 107) were injected into the mice, and their migration to Peyer's patches was observed. Bar represents 100 μm. Gastroenterology 2002 122, 734-744DOI: (10.1053/gast.2002.31899) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 (A) Time-course changes in the number of T lymphocytes sticking to postcapillary venules of Peyer's patches and the difference between SPLs and LPLs. Lymphocytes located inside or along the venules were counted in a 1-mm2 observation field. #P < 0.05, compared with SPLs. Values are means ± SEM for 6 separate animals. (B) Effect of monoclonal antibodies against adhesion molecules on the sticking of SPLs to postcapillary venules of Peyer's patches 20 minutes after infusion. SPLs were treated with a monoclonal antibody (100 μg/mL) against L-selectin (MEL-14), α4-integrin (PS/2), and β7-integrin (FIB27) before infusion. In some experiments, the animals were pretreated (30 minutes before lymphocyte infusion) with a monoclonal antibody against MAdCAM-1 (2 mg/kg). #P < 0.05, compared with controls. Values are means ± SEM for 6 separate animals. Gastroenterology 2002 122, 734-744DOI: (10.1053/gast.2002.31899) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Representative photomicrographs showing the distribution of CFSE-labeled lamina propria T lymphocytes (LPLs) (left) and splenic T lymphocytes (SPLs) (right) in microvessels of the villus tips of intestinal mucosa. Villus tip capillaries were observed from the mucosal side. SPLs and LPLs (1 × 107) were injected into the mice, and their migration to villus microvessels was observed. Bar represents 100 μm. Gastroenterology 2002 122, 734-744DOI: (10.1053/gast.2002.31899) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) Time-course changes in the number of T lymphocyte sticking to microvessels in the villus tips and the difference between SPLs and LPLs. #P < 0.05, compared with SPLs. Values are means ± SEM for 6 separate animals. (B) Effect of monoclonal antibodies against adhesion molecules on the sticking of LPLs to microvessels in the villus tips 20 minutes after infusion. LPLs were treated with a monoclonal antibody (100 μg/mL) against L-selectin (MEL-14), αE-integrin (M290), α4-integrin (PS/2), β7-integrin (FIB27), and CD11a (M17/4) before infusion. In some experiments, the animals were pretreated (30 minutes before lymphocyte infusion) with a monoclonal antibody against MAdCAM-1 (2 mg/kg, MECA-367, either in whole or in F(ab')2 fragments). A functionally nonblocking anti–MAdCAM-1 mAb (MECA-89) was also used under the same conditions. #P < 0.05, compared with controls. Values are means ± SEM for 6 separate animals. Gastroenterology 2002 122, 734-744DOI: (10.1053/gast.2002.31899) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Representative image of the distribution of MAdCAM-1 expression in small intestinal villi as determined by immunohistochemistry. The primary antibodies used in the immunostaining were monoclonal antibodies that react to MAdCAM-1 (MECA367). These sections were treated with biotinylated anti-rat IgG and visualized by streptavidin-FITC. MAdCAM-1 was mainly expressed in the endothelium of venules in the lamina propria of intestinal mucosa just above the muscularis mucosa, but significant MAdCAM-1 expression was also observed in the microvessels of villus projections. Gastroenterology 2002 122, 734-744DOI: (10.1053/gast.2002.31899) Copyright © 2002 American Gastroenterological Association Terms and Conditions