1. Volume 133, Issue 1, Pages 124-136 (July 2007)
Inducible IL-12-Producing B Cells Regulate Th2-Mediated Intestinal Inflammation 
Ken Sugimoto, Atsuhiro Ogawa, Yasuyo Shimomura, Kiyotaka Nagahama, Atsushi Mizoguchi, Atul K. Bhan 
Gastroenterology 
Volume 133, Issue 1, Pages (July 2007)
DOI: /j.gastro
Copyright © 2007 AGA Institute Terms and Conditions

2. Figure 1
Significant up-regulation of IL-12p35 expression in the MLN of TCRαKO mice but not αμDKO or αIL10DKO mice. (A) RPA was performed using total RNA from MLN of WT, TCRαKO, αμDKO, and αIL10DKO mice without (−) or with (+) colitis. (B) RPA was preformed using total RNA from colonic LP of nondiseased WT mice and diseased TCRαKO, αμDKO, and αIL10DKO mice. (C) Total RNA was isolated from the MLN and the colonic LP of WT, TCRαKO, αμDKO, and αIL10DKO mice and subjected to real-time PCR using specific primers for the detection of IL-12p35 and β-actin mRNA. The data represent the average of the values of 2-CT of IL-12p35/2-CT of β-actin (n = 3) ± SEM.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
Involvement of IL-10-producing B cells in MLN IL-12p35 expression under a setting of colonic inflammation. (A) Total RNA was isolated from the MLN of WT, TCRαKO, and αIL10DKO mice and subjected to real-time PCR using specific primers for the detection of IL-10 and β-actin mRNA. The data represent the average of the values of 2-CT of IL-10/2-CT of β-actin (n = 3) ± SEM. N.D. indicates “not detectable.” (B) Total RNA was isolated from the MLN of WT and αμDKO mice without or with transfer of B cells from WT, TCRαKO, or αIL10DKO mice and then analyzed by RPA for the detection of IL-12p35 and p40. (C) Total RNA were isolated from the MLN of αIL10DKO and αμIL10TKO mice treated without or with recombinant IL-10 (2 mg) and then analyzed by RPA for the detection of IL-12p35 and p40.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
IL-12p70 production by MLN B cells under intestinal inflammatory condition. (A) MLN cells from TCRαKO mice were incubated with either FITC or PE-conjugated anti-CD4, anti-Igμ, anti-CD11c, anti-F4/80, anti-Gr-1, and anti-pan-NK cell mAbs and the stained cells positively sorted after subsequent incubation with anti-FITC or PE magnetic beads under a magnetic cell sorting (MACS) system. RNA extracted from the purified cell subsets (5 × 105 cells) was subjected to real-time PCR using the specific primers for detection of IL-12p35 and β-actin. The data represent the average of the values of 2-CT of IL-12p35/2-CT of β-actin (n = 3) ± SEM. (B) Purified B220+ B cells (1.5 × 105) from the MLN of WT or TCRαKO mice were cultured for 48 hours without (cont) or with peptidoglycan (PEG, 10 μg/mL), Pam3CSK4 (Pam, 1 μg/mL), poly (IC) (25 μg/mL), LPS (0.2 μg/mL), flagellin (1 μg/mL), ssRNA40 (10 μg/mL), or CpG (1 μmol/L). In addition, based on the result, CpG stimulation was performed with an additional stimulation with BCR ligation (50 μg/mL Igμ Ab) (CpG + BCR). The culture supernatants were then subjected to ELISA for the detection of IL-12p70 (p35/p40). The data represent the averages (n = 6) ± SEM. (C) Total MLN cells (1 × 106) from TCRαKO mice were cultured without (No stimulation) or with F(ab’)2 goat anti-mouse Igμ (BCR ligation, 50 μg/mL), CpG (1 μmol/L) alone, or F(ab’)2 goat anti-mouse Igμ (50 μg/mL) plus CpG (1 μmol/L) for 15 hours. After cytoplasmic staining of IL-12p70 and surface staining of CD19 and B220, the cells were immediately analyzed by FACS. The expressions of IL-12p70 vs CD19 on the gated B220+ cells are shown. (D) Total MLN cells (1 × 106) from TCRαKO or αIL10DKO mice were cultured with CpG (1 μmol/L) for 15 hours. After cytoplasmic staining of IL-12p70 and surface staining of CD19 and B220, the cells were immediately analyzed by FACS. The expressions of IL-12p70 vs CD19 on the gated B220+ cells are shown. (E) Total MLN cells (1 × 106) from TCRαKO mice were cultured with CpG (5 μmol/L) for 15 hours. The cells were then subjected to cytoplasmic staining for IL-12p70 and surface staining for CD19, CD3ε, CD11c, or F4/80 and immediately analyzed by FACS. To increase the number of dendritic cells and macrophages, collagenase treatment was performed for the isolation of cells shown in the bottom 2 panels. (F) Total MLN cells (1 × 106) from TCRαKO mice were cultured with CpG (5 μmol/L) for 15 hours. The cells were then subjected to cytoplasmic staining for IL-12p70 and IL-10 and surface staining for CD19 and immediately analyzed by FACS. The expressions of IL-12p70 vs IL-10 on the gated CD19+ cells are shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
Exacerbation of intestinal inflammation in TCRαKO mice in the absence of IL-12p35. (A and B) Gross features of the large intestine of TCRαKO (A) and αp35DKO (B) mice at 24 weeks of age are shown. (C and D) Histologic findings (×10 objective) of distal colon of TCRαKO (C) and αp35DKO (D) mice at 24 weeks of age are shown. (E) Disease scores (0–9) of colitis in 22- to 25-week-old TCRαKO (n = 41) and αp35DKO (n = 38) mice as assessed by both gross and histologic findings are summarized. Each dot represents an individual mouse. There is a statistically significant difference in the disease scores between TCRαKO and αp35DKO mice (P < .05).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
Up-regulation of IL-17A and IL-17F in the colonic LP of αp35DKO mice as compared with TCRαKO mice. (A) Total RNA was isolated from colonic LP without epithelial cells of WT, TCRαKO, or αp35DKO mice without (−) or with (+) colitis. Expression levels of IFN-γ, IL-4, IL-17A, and IL-17F mRNA as assessed by real-time PCR are shown. The data represent the average of the values of 2-CT of target molecule/2-CT of β-actin (n = 3) ± SEM. (B) Total RNA were isolated from 3 × 105 purified colonic LP CD4+ T cells of WT, TCRαKO, and αp35DKO mice and then subjected to real-time PCR for detection of IL-17A, IL-17F, and β-actin. The data represent the average of the values of 2-CT of target molecule/2-CT of β-actin (n = 3) ± SEM. (C) Purified CD4+ T cells from the colon of TCRαKO and αp35DKO mice were stimulated with anti-CD3ε mAb/anti-CD28 mAb for 48 hours. The culture supernatants were subjected to ELISA for detection of IL-17A. The data represent the average ± SEM of triplicate data from 3 mice of each group. (D) Total RNA was isolated from colonic LP and MLN of WT, TCRαKO, and αp35DKO mice and then subjected for detection of p19 and β-actin. The data represent the average of the values of 2-CT of target molecule/2-CT of β-actin (n = 3) ± SEM.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
An ability of IL-12p35-producing B cells to attenuate chronic colitis. (A–C) The diameter of the distal colon was measured by opening the abdomen of anesthetized αμDKO mice at 15 weeks of age (upper panels in A and B). Fourteen days after the operation, the αμDKO mice were repeatedly reconstituted with purified MLN B cells (1.5 × 107, every 10 days, 3 times) from TCRαKO mice (A) or αp35DKO mice (B). The recipient mice were killed on day 10 after the final B-cell transfer (bottom panels in A and B). The gross findings of distal colon before (upper panels in A and B) and after (bottom panels in A and B) the B-cell transfer are shown. The numbers in each panel represent the colonic diameter. The averages of results (TCRα > αμ; n = 8, αp35 > αμ; n = 9) are summarized in panel C. (D) Disease score of αμDKO mice after transfer of MLN B cells from TCRαKO mice or αp35DKO mice are shown. Each dot represents an individual mouse. (E) Total RNA were isolated from 3 × 105 purified colonic LP CD4+ T cells from αμDKO mice reconstituted with MLN B cells from TCRαKO mice or αp35DKO mice and then subjected to real-time PCR for detection of IL-17A, IL-17F, and β-actin. The data represent the average of the values of 2-CT of target molecule/2-CT of β-actin (n = 3) ± SEM.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

8. Figure 7
Neutralization of IL-12p40 activity inhibits the B-cell-mediated attenuation of colitis. (A–C) The diameter of the distal colon was measured by opening the abdomen of anesthetized αμDKO mice at 15 weeks of age (upper panels in A and B). Fourteen days after the operation, the αμDKO mice were repeatedly reconstituted with purified MLN B cells (1.5 × 107, every 10 days, 3 times) from TCRαKO mice. The recipient mice were treated with anti-IL-12p40 mAb (C17.8, 1 mg/injection, every 10 days, 3 times) (B) or control Ig (A). The recipient mice were killed on day 10 after the final B-cell transfer (bottom panels in A and B). The gross findings of distal colon before (upper panels in A and B) and after (bottom panels in A and B) the B-cell transfer are shown. The numbers in each panel represent the colonic diameter. The averages of results (n = 3) are summarized in panel C. (D) Histologic findings of the distal part of colon of αμDKO mice that are reconstituted with B cells from TCRαKO mice and received treatment with anti-IL-12p40 mAb or control Ig are shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions