Volume 122, Issue 7, Pages 2001-2010 (June 2002) Critical role of interleukin 5 and eosinophils in concanavalin A–induced hepatitis in mice  Hubert Louis, Alain Le Moine, Véronique Flamand, Nathalie Nagy, Eric Quertinmont, Frédéric Paulart, Daniel Abramowicz, Olivier Le Moine, Michel Goldman, Jacques Devière  Gastroenterology  Volume 122, Issue 7, Pages 2001-2010 (June 2002) DOI: 10.1053/gast.2002.33620 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Critical involvement of IL-5 in Con A–induced hepatitis. C57BL/6 WT or IL-5−/− mice were injected with 0.3 mg Con A. (A) Serum ALT values (black bars: WT mice; white bars: IL-5−/− mice), (B) rectal temperature, and (C) glycemia (black circles: WT mice; white circles: IL-5−/− mice) were measured at different time-points (mean ± SEM of 10 mice per time-point, *P = 0.02 at 8 hours, P = 0.004 at 14 hours, and P = 0.003 at 24 hours for ALT; P < 0.02 for rectal temperature and P < 0.01 for glycemia). Results are representative of 3 independent experiments. (D) Mortality was studied after 0.75 mg Con A administration (black circles: WT mice; white circles: IL-5−/− mice) (P = 0.02, log rank test, n = 18 per group of mice). Gastroenterology 2002 122, 2001-2010DOI: (10.1053/gast.2002.33620) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Liver histology 8 hours after Con A injection in (A) C57BL/6 WT mice, (B) IL-5−/− mice, (C) control MAb-injected C57BL/6 WT mice, and (D) eosinophil-depleted WT mice through anti-CCR3 MAb injections (H&E, original magnification 100×). Liver eosinophil infiltrate (black arrows) is shown (E and G) 8 hours after Con A injection in C57BL/6 WT mice, (F and H) but not in IL-5−/− mice. (E and F, cyanide-resistant eosinophil peroxidase activity; G and H, immunostaining with CCR3-specific anti-serum; original magnification 400×). Gastroenterology 2002 122, 2001-2010DOI: (10.1053/gast.2002.33620) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Cytokine profiles and IL-5 mRNA expression after Con A injection. (A) Serum levels of IL-5, eotaxin, IL-4, TNF-α, IFN-γ, and IL-10 were measured in C57BL/6 WT, IL-5−/−, and β2 m−/− mice (for β2 m−/− mice, measurements were performed at 0, 2, 5, and 8 hours for IL-5 and at 0, 2, and 8 hours for IL-4, TNF-α, and IFN-γ) (mean ± SEM of 5–10 mice per time-point, *P < 0.02 for IL-4, P < 0.03 for IFN-γ, and P < 0.03 for IL-10. ND means not done). (B) Kinetic study for IL-5 mRNA in C57BL/6 WT mice. Real-time PCR was performed for IL-5. Results are expressed in mRNA copy numbers calculated relative to β-actin copy numbers. The mean ± SEM of 3 mice per time-point is represented. Gastroenterology 2002 122, 2001-2010DOI: (10.1053/gast.2002.33620) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Serum ALT levels 8 hours after Con A injection in various groups of mice. (A) C57BL/6 WT or Fas−/− (lpr/lpr) mice were injected with anti–IL-5 (□) or control (▨) mAbs 2 hours before Con A challenge. *P < 0.01 between WT mice; P < 0.02 between Fas−/− mice (mean ± SEM of 6–13 mice per group). (B) C57BL/6 WT or IL-5−/− mice were injected with the anti–IL-10 mAb (□) or control mAb (▨) 2 hours before Con A challenge (mean ± SEM of 6 mice per group). (C) C57BL/6 WT mice were injected with anti-CCR3 (□) or control MAb (▨) on day −3, −1, and 1 hour before Con A challenge. P < 0.02 (mean ± SEM of 9 mice per group). Gastroenterology 2002 122, 2001-2010DOI: (10.1053/gast.2002.33620) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Con A–induced hepatitis depends on NKT cell–derived IL-5 production. (A) IL-4 and IL-5 production were measured in vitro on culture supernatants of NK1.1+ MACS-enriched or depleted spleen cell populations obtained either from WT or IL-5−/− mice (mean values in triplicate samples with SEM of 2 separate experiments, *P = 0.002, IL-5−/− NK1.1 enriched vs. WT NK1.1 enriched; **P = 0.00002, WT NK1.1 depleted vs. WT NK1.1 enriched; ***P = 0.02, WT NK1.1 depleted vs. WT NK1.1 enriched). □, WT; ▨, IL-5−/−. (B) NK1.1+ MACS-enriched spleen cells obtained either from WT or IL-5−/− mice, or NK1.1+-depleted spleen cells obtained from WT mice were injected intrasplenically into β2m−/− mice 1 hour before Con A injection. Serum ALT levels were measured 8 hours later (mean ± SEM of 6–8 mice per group, *P < 0.02, as compared with WT NK1.1 enriched). Gastroenterology 2002 122, 2001-2010DOI: (10.1053/gast.2002.33620) Copyright © 2002 American Gastroenterological Association Terms and Conditions