Volume 134, Issue 1, Pages 292-305 (January 2008) Disordered Pancreatic Inflammatory Responses and Inhibition of Fibrosis in CD39-Null Mice  Beat M. Künzli, Philipp Nuhn, Keiichi Enjyoji, Yara Banz, Rex N. Smith, Eva Csizmadia, Detlef Schuppan, Pascal O. Berberat, Helmut Friess, Simon C. Robson  Gastroenterology  Volume 134, Issue 1, Pages 292-305 (January 2008) DOI: 10.1053/j.gastro.2007.10.030 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Assessment of pancreatitis. (A) After 6 weeks of CIP, significant decreases in pancreas weights of wt vs CD39-null mice were observed (secondary to atrophy of the pancreas, *P < .005). (B) Saline injection for 6 weeks did not affect pancreas weights (sham treatment). (C and D) Blood amylase activity revealed no differences between cerulein- or sham-treated wt and CD39-null mice (normal range of used test, up to 100 IU/L). (E) Heightened levels of IFN-γ were detected only in sera from CD39-null mice after 3 weeks of pancreatitis induction (*P < .05). (F) Blood sugar levels remained normal and did not differ between the treatment groups. Gastroenterology 2008 134, 292-305DOI: (10.1053/j.gastro.2007.10.030) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Western analyses. Protein expression was determined from baseline pancreas without treatment, after 3 and 6 weeks of CIP. (A) Representative samples showing high induction of CD39 at 3 and 6 weeks of CIP in wt mice. CD39-null mice show increased protein expression of CD39L1 and P2X7 (at 3 wk). P2Y2 was up-regulated after 6 weeks. MMP-2 was up-regulated significantly but only in CD39-null mice. TGF-β1 was equally up-regulated at 3 weeks of CIP. CD45 was expressed predominantly in CD39-null mice at 3 weeks of CIP treatment. (A) Fibronectin was highly up-regulated at 6 weeks’ of treatment, and more prominently expressed in wt mice. Each lane (40 μg total protein/lane) represents 1 pancreas sample. Shown is 1 representative individual mouse per group (baseline pancreas, CIP at 3 and 6 weeks’ time, respectively). (B) Protein lysates from primary cultured PSCs showed high expression for CD39. P2X7 expression was more prominent in wt cells when compared with CD39-null PSCs. Protein levels of fibronectin were increased in wt PSCs vs CD39-null PSCs. Gastroenterology 2008 134, 292-305DOI: (10.1053/j.gastro.2007.10.030) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Pancreas morphology. (A) Pancreas architecture in sham-treated remained unchanged in wt and CD39-null mice. (B) Panels show representative Masson’s trichrome staining sections from wt and CD39-null mice. Histologic parameters such as (C) fibrosis (P < .02) and (D) atrophy (*P < .02) revealed significant differences between wt and CD39-null pancreases. Gastroenterology 2008 134, 292-305DOI: (10.1053/j.gastro.2007.10.030) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Immunohistochemistry in CIP. Panels show representative immunohistochemical analyses of CD39 and P2X7 in tissues of wt and CD39-null mice after 6 weeks of CIP. (A and B) Control pancreas samples from sham-treated wt and CD39-null mice did not reveal any significant architectural changes (nor alterations in cD39 expression in wt) after saline treatment. (B and D) There was no reactivity for CD39 in CD39-null mice. (C) With pancreatitis, the density of CD39-positive staining increased in wt samples and was localized to endothelial and other cells in the interacinar and interseptal spaces (Figure 3C, insert). These cells share homologies with myofibroblast-like cells and resemble PSCs in localization and morphology. (E and F) P2X7 is localized mainly to immune cells (lc, leukocyte) and elements of the vasculature in these biopsies of CP. Gastroenterology 2008 134, 292-305DOI: (10.1053/j.gastro.2007.10.030) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Immunohistochemistry and immunofluorescence in CP. (A and B) CD34, a myofibroblast and stromal cell marker localized within the periacinar and periductular areas. CD39 is colocalized with CD34. (C and D) The double-positive cells display a distinct morphology, are single cells with spindle-like protrusions, and comparable with the described phenotype and morphology of PSCs. (E and F) CD39L1 was colocalized to CD34 in the rarely encountered areas of dense fibrosis in CD39-null mice (Figure 4F, insert), whereas in dense fibrotic areas of wt mice, colocalization was only minimal. Gastroenterology 2008 134, 292-305DOI: (10.1053/j.gastro.2007.10.030) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Phenotype and differentiation of PSCs in primary cultures. (A and B) CD39-null PSCs displayed a highly significant proliferation deficit in primary culture compared with wt PSCs (P < .02). (A) Stimulation of CD39-null PSCs with PDGF (5 ng/mL) did not lead to increased proliferation in CD39-null PSCs, whereas wt PSCs responded to PDGF. (C) Increased concentration of ATP levels (10 and 50 μmol but not 150 and 400 μmol) in culture media significantly increased proliferation of wt PSCs (P < .05). (D) Significant differences in mRNA expression profile for CD39 and P2X7 in cultures of wt PSCs compared with CD39-null PSCs. CD39-null PSCs were viable and differentiated in a comparable manner with wt PSCs, as indicated by equal expression of α-SMA on (E) mRNA and (F) morphologically. Morphology of PSCs revealed high positive staining for α-SMA and desmin in cultured PSCs. (F) Differentiation and viability were comparable in wt and CD39-null PSCs. (D and E) ■, wt; □, CD39-null. Gastroenterology 2008 134, 292-305DOI: (10.1053/j.gastro.2007.10.030) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Proliferation defect in CD39-null PSCs. In general, a lack of plasticity was noted in CD39-null PSC responses to growth factors and TGF-β. wt and CD39-null PSCs were stimulated with PDGF (5 ng/mL) or TGF-β1 (2 ng/mL). (A) The mRNA expression profiles reached moderate levels of CD39 expression in wt PSCs (*P < .03). (B) CD39L1 levels of expression in wt (and to a lesser extent in CD39-null PSCs) decreased when stimulated with PDGF or TGF-β1 (*P < .01). P2X7 expression in wt PSCs was significantly increased when compared with CD39-null PSCs (*P < .03). P2X7 levels trended lower during stimulation with PDGF and TGF-β1. (D) Levels of P2Y2 were comparable during stimulation experiments. (E) At baseline conditions and under stimulation with PDGF, α-SMA was expressed at comparable levels in wt and mutant mice. (F) Procollagen-α1 production was decreased significantly basally in CD39-null PSCs (*P < .03). (F) Most strikingly, procollagen-α1 expression in wt PSCs increased significantly when stimulated with the profibrogenic cytokine TGF-β1 (*P < .03). This defect suggests nonresponsiveness of and probable defects in collagen production in the CD39-null PSCs. Gastroenterology 2008 134, 292-305DOI: (10.1053/j.gastro.2007.10.030) Copyright © 2008 AGA Institute Terms and Conditions