1. Volume 117, Issue 3, Pages 536-545 (September 1999)
Functional expression of costimulatory molecule CD86 on epithelial cells in the inflamed colonic mucosa 
Atsushi Nakazawa, Mamoru Watanabe, Takanori Kanai, Tomoharu Yajima, Motomi Yamazaki, Haruhiko Ogata, Hiromasa Ishii, Miyuki Azuma, Toshifumi Hibi 
Gastroenterology 
Volume 117, Issue 3, Pages (September 1999)
DOI: /S (99)
Copyright © 1999 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Immunohistochemical staining of CD86 and CD80 in human colonic mucosal tissues. Frozen sections of colonic mucosal tissues from patients with UC and normal controls (NL) were stained with (A) anti-CD86 or (B) anti-CD80. Four representative stainings for CD86 and 1 for CD80 from 25 tissues with UC and 20 normal controls are shown (bar = 50 μm).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

3. Fig. 2
(A) Flow cytometric analysis of CD86 and CD80 expression on freshly isolated colonic epithelial cells in patients with UC and normal control (NL) subjects. Freshly isolated colonic epithelial cells from UC and NL subjects were stained with either anti-CD86 or anti-CD80, followed by biotinylated anti-mouse IgG and streptavidin-PE. Samples were analyzed by flow cytometry. Epithelial cells were identified by characteristic forward-angle and side-scatter profiles. Data are displayed as histograms (bold lines) with the Ig control histograms (solid lines). ΔMFI from each of 5 subjects for UC and NL was indicated as the mean ΔMFI ± SD. (B) CD86 expression on B9+ epithelial cells by 2-color flow cytometric analysis. Freshly isolated colonic epithelial cells from UC and NL subjects were stained with PE-conjugated anti-CD86 MAb and biotinylated anti–B9 MAb, followed by streptavidin-FITC. Samples were analyzed by flow cytometry. Data are displayed as dot plots, and quadrant markers are positioned to include >98% of control Ig-stained cells in the lower left. Representative data from each of 3 UC and NL subjects are shown.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

4. Fig. 2
(A) Flow cytometric analysis of CD86 and CD80 expression on freshly isolated colonic epithelial cells in patients with UC and normal control (NL) subjects. Freshly isolated colonic epithelial cells from UC and NL subjects were stained with either anti-CD86 or anti-CD80, followed by biotinylated anti-mouse IgG and streptavidin-PE. Samples were analyzed by flow cytometry. Epithelial cells were identified by characteristic forward-angle and side-scatter profiles. Data are displayed as histograms (bold lines) with the Ig control histograms (solid lines). ΔMFI from each of 5 subjects for UC and NL was indicated as the mean ΔMFI ± SD. (B) CD86 expression on B9+ epithelial cells by 2-color flow cytometric analysis. Freshly isolated colonic epithelial cells from UC and NL subjects were stained with PE-conjugated anti-CD86 MAb and biotinylated anti–B9 MAb, followed by streptavidin-FITC. Samples were analyzed by flow cytometry. Data are displayed as dot plots, and quadrant markers are positioned to include >98% of control Ig-stained cells in the lower left. Representative data from each of 3 UC and NL subjects are shown.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

5. Fig. 3
(A) CD86 and (B) CD80 mRNA expression of freshly isolated colonic epithelial cells. Analysis of RT-PCR–amplified transcripts of CD80, CD86, and GPDH (lower panels) at 30, 35, and 40 cycles from cells of colonic mucosa from normal control (lane 1), UC patients (lane 2), and Raji cells as a positive control (lane 3). Lane 4 shows a negative control. The predicted product size for CD86, CD80, and GPDH is 833, 804, and 983 bp, respectively. (C) Comparative expression for CD86 mRNA in freshly isolated colonic epithelial cells between UC and NL subjects. PCR products for CD86 mRNA at 40 cycles from colonic mucosa from 7 UC and 7 NL subjects are shown.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

6. Fig. 3
(A) CD86 and (B) CD80 mRNA expression of freshly isolated colonic epithelial cells. Analysis of RT-PCR–amplified transcripts of CD80, CD86, and GPDH (lower panels) at 30, 35, and 40 cycles from cells of colonic mucosa from normal control (lane 1), UC patients (lane 2), and Raji cells as a positive control (lane 3). Lane 4 shows a negative control. The predicted product size for CD86, CD80, and GPDH is 833, 804, and 983 bp, respectively. (C) Comparative expression for CD86 mRNA in freshly isolated colonic epithelial cells between UC and NL subjects. PCR products for CD86 mRNA at 40 cycles from colonic mucosa from 7 UC and 7 NL subjects are shown.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

7. Fig. 3
(A) CD86 and (B) CD80 mRNA expression of freshly isolated colonic epithelial cells. Analysis of RT-PCR–amplified transcripts of CD80, CD86, and GPDH (lower panels) at 30, 35, and 40 cycles from cells of colonic mucosa from normal control (lane 1), UC patients (lane 2), and Raji cells as a positive control (lane 3). Lane 4 shows a negative control. The predicted product size for CD86, CD80, and GPDH is 833, 804, and 983 bp, respectively. (C) Comparative expression for CD86 mRNA in freshly isolated colonic epithelial cells between UC and NL subjects. PCR products for CD86 mRNA at 40 cycles from colonic mucosa from 7 UC and 7 NL subjects are shown.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

8. Fig. 4
Flow cytometric analysis of CD86 and CD80 expression on colonic epithelial cell line HT29-18-N2 after IFN-γ treatment (bold lines). Cells were stained with anti-CD86 and anti-CD80 MAbs, followed by biotinylated anti-mouse IgG antibody and streptavidin-PE. Solid lines show the profiles of cells stained with an isotype-matched antibody for a negative control. Raji cells were used as positive control for CD86 and CD80 staining. For CD86 expression, ΔMFI in IFN-γ–treated HT29-18-N2 cells (8.16 ± 3.03) was significantly (P < 0.05) increased compared with that in unstimulated HT29-18-N2 cells (0.39 ± 2.84). In contrast, ΔMFI in IFN-γ–treated HT29-18-N2 cells (1.89 ± 1.80) was not increased compared with that in unstimulated HT29-18-N2 cells (2.13 ± 3.01) for CD80 expression.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

9. Fig. 5
(A) CD86 and (B) CD80 mRNA expression in a colonic epithelial cell line HT29-18-N2. Semiquantitative PCR analyses at 30, 35, and 40 cycles from HT29-18-N2 cells unstimulated (lane 1), IFN-γ–stimulated for 48 hours (lane 2), and Raji cells (lane 3) were performed as described in Figure 3.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

10. Fig. 5
(A) CD86 and (B) CD80 mRNA expression in a colonic epithelial cell line HT29-18-N2. Semiquantitative PCR analyses at 30, 35, and 40 cycles from HT29-18-N2 cells unstimulated (lane 1), IFN-γ–stimulated for 48 hours (lane 2), and Raji cells (lane 3) were performed as described in Figure 3.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

11. Fig. 6
(A) Determination of a suboptimal dose of PHA. Purified CD4+ T cells were cultured in the presence of titrated amounts of PHA for 72 hours. Cultures were pulsed for a final 18 hours, and incorporation for [3H]thymidine was measured. Results from 3 individuals are presented. (B) CD4+ T-cell proliferation costimulated with colonic epithelial cells. Purified CD4+ T cells (1 × 105 cells/well) were cocultured with an indicated number of mitomycin-C–treated IFN-γ–stimulated (○) or unstimulated (□) HT29-18-N2 cells in the presence of PHA (1:5000) for 72 hours, and proliferative responses were measured as described previously. IFN-γ–treated HT29-18-N2 cells failed to stimulate CD4+ T cells in mixed culture in the absence of PHA (◊). The count for CD4+ T-cell culture with PHA alone is always <3000 cpm. Data are representative of 3 independent experiments. Each value shows the mean ± SD from triplicate culture. (C) Inhibitory effects on CD4+ T-cell proliferative responses costimulated with IFN-γ–treated HT29-18-N2 cells. CD4+ T cells were cocultured with IFN-γ–treated HT29-18-N2 cells (1 × 104 cells/well) in the presence of PHA (1:5000) for 72 hours. Blocking MAb against either CD80, CD86, ICAM-1, CD4, or HLA-DR was added at the start of the assay at a final concentration of 5–10 μg/mL. Values are the mean ± SD from 5 experiments using different donors. *Statistically significantly different (P < 0.01).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

12. Fig. 6
(A) Determination of a suboptimal dose of PHA. Purified CD4+ T cells were cultured in the presence of titrated amounts of PHA for 72 hours. Cultures were pulsed for a final 18 hours, and incorporation for [3H]thymidine was measured. Results from 3 individuals are presented. (B) CD4+ T-cell proliferation costimulated with colonic epithelial cells. Purified CD4+ T cells (1 × 105 cells/well) were cocultured with an indicated number of mitomycin-C–treated IFN-γ–stimulated (○) or unstimulated (□) HT29-18-N2 cells in the presence of PHA (1:5000) for 72 hours, and proliferative responses were measured as described previously. IFN-γ–treated HT29-18-N2 cells failed to stimulate CD4+ T cells in mixed culture in the absence of PHA (◊). The count for CD4+ T-cell culture with PHA alone is always <3000 cpm. Data are representative of 3 independent experiments. Each value shows the mean ± SD from triplicate culture. (C) Inhibitory effects on CD4+ T-cell proliferative responses costimulated with IFN-γ–treated HT29-18-N2 cells. CD4+ T cells were cocultured with IFN-γ–treated HT29-18-N2 cells (1 × 104 cells/well) in the presence of PHA (1:5000) for 72 hours. Blocking MAb against either CD80, CD86, ICAM-1, CD4, or HLA-DR was added at the start of the assay at a final concentration of 5–10 μg/mL. Values are the mean ± SD from 5 experiments using different donors. *Statistically significantly different (P < 0.01).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

13. Fig. 6
(A) Determination of a suboptimal dose of PHA. Purified CD4+ T cells were cultured in the presence of titrated amounts of PHA for 72 hours. Cultures were pulsed for a final 18 hours, and incorporation for [3H]thymidine was measured. Results from 3 individuals are presented. (B) CD4+ T-cell proliferation costimulated with colonic epithelial cells. Purified CD4+ T cells (1 × 105 cells/well) were cocultured with an indicated number of mitomycin-C–treated IFN-γ–stimulated (○) or unstimulated (□) HT29-18-N2 cells in the presence of PHA (1:5000) for 72 hours, and proliferative responses were measured as described previously. IFN-γ–treated HT29-18-N2 cells failed to stimulate CD4+ T cells in mixed culture in the absence of PHA (◊). The count for CD4+ T-cell culture with PHA alone is always <3000 cpm. Data are representative of 3 independent experiments. Each value shows the mean ± SD from triplicate culture. (C) Inhibitory effects on CD4+ T-cell proliferative responses costimulated with IFN-γ–treated HT29-18-N2 cells. CD4+ T cells were cocultured with IFN-γ–treated HT29-18-N2 cells (1 × 104 cells/well) in the presence of PHA (1:5000) for 72 hours. Blocking MAb against either CD80, CD86, ICAM-1, CD4, or HLA-DR was added at the start of the assay at a final concentration of 5–10 μg/mL. Values are the mean ± SD from 5 experiments using different donors. *Statistically significantly different (P < 0.01).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

14. Fig. 7
(A) CD4+ T-cell proliferative responses costimulated with freshly isolated colonic epithelial cells. Purified CD4+ T cells (1 × 105 cells/well) were cocultured with or without freshly isolated colonic epithelial cells (1 × 104 cells/well) from UC and NL subjects in the presence of PHA (1:5000) for 72 hours, and proliferative responses were measured as described previously. Each value shows the mean ± SD from triplicate cultures. (B) Inhibitory effects on CD4+ T-cell proliferative responses costimulated with freshly isolated colonic epithelial cells from patients with UC. Purified CD4+ T cells were cocultured with freshly isolated colonic epithelial cells from UC patients (1 × 104 cells/well) in the presence of PHA (1:5000) for 72 hours. Blocking MAbs were added as described in Figure 6C. Values are the mean ± SD from 3 patients with UC. *Statistically significantly different (P < 0.01).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

15. Fig. 7
(A) CD4+ T-cell proliferative responses costimulated with freshly isolated colonic epithelial cells. Purified CD4+ T cells (1 × 105 cells/well) were cocultured with or without freshly isolated colonic epithelial cells (1 × 104 cells/well) from UC and NL subjects in the presence of PHA (1:5000) for 72 hours, and proliferative responses were measured as described previously. Each value shows the mean ± SD from triplicate cultures. (B) Inhibitory effects on CD4+ T-cell proliferative responses costimulated with freshly isolated colonic epithelial cells from patients with UC. Purified CD4+ T cells were cocultured with freshly isolated colonic epithelial cells from UC patients (1 × 104 cells/well) in the presence of PHA (1:5000) for 72 hours. Blocking MAbs were added as described in Figure 6C. Values are the mean ± SD from 3 patients with UC. *Statistically significantly different (P < 0.01).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions