1. Volume 132, Issue 4, Pages 1504-1514 (April 2007)
Biliary Epithelial Cell Antibodies Link Adaptive and Innate Immune Responses in Primary Sclerosing Cholangitis 
Azza Karrar, Ulrika Broomé, Towe Södergren, Marie Jaksch, Annika Bergquist, Mikael Björnstedt, Suchitra Sumitran–Holgersson 
Gastroenterology 
Volume 132, Issue 4, Pages (April 2007)
DOI: /j.gastro
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2. Figure 1
(A) Human BECs exhibited typical epithelial cell morphology when grown in epithelial cell selective medium. (Original magnification 20× and 40×.) (B) A representative picture (n = 3) of flow cytometric analysis showing that isolated BECs stained positive for cytokeratin 7 (gray line) and cytokeratin 19 (filled line) but stained negative with Ab to a fibroblast marker, smooth muscle cells marker α-actin, von Willebrand factor (gray line), and vascular cell adhesion molecule (filled line). A negative control using only secondary Ab was also included (black line). (C) A representative picture (n = 3) of flow cytometric analysis showing the strong binding of PSC whole IgG fraction (0.5 mg/mL) and PSC IgG F(ab′)2 fraction (2 mg/mL) to BECs but not to primary human renal epithelial cells, lung epithelial cells, or lymphocytes. Normal IgG (0.5 mg/mL) or IgG from PSC negative for BEC-Ab did not bind to any of the cells tested (filled histogram and dotted line, respectively). Secondary Ab was used as negative control (black line). (D) Immunocytochemical analysis confirmed the binding of PSC IgG to BECs. (a) Unstimulated and (b) normal IgG-stimulated BECs did not show any binding to BECs, while stimulation with PSC (c) whole and (d) F(ab′)2 IgG showed an intense positive staining (red/brown staining). (Original magnification 40×).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
(A) Immunofluorescent staining of human BECs showed expression of TLR4 (green, FITC) and TLR9 (green, FITC) on BECs treated with PSC IgG. The cells were double stained for cytokeratin 19 (red, Cy3). The nucleus is stained blue (4′,6-diamidino-2-phenylindole). In some areas, the TLRs and cytokeratin 19 were colocalized (yellow). (B) A representative picture (n = 3) of flow cytometric analysis showing that PSC whole and F(ab′)2 IgG fractions did not induce protein expression for TLR1, TLR2, and TLR3 but up-regulated expression of TLR4, TLR9, and the adapter signaling molecule MyD88 in BECs.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
(A) Human BECs were either left untreated or preincubated with PSC IgG (0.5 mg/mL, 20 minutes) or normal IgG (0.5 mg/mL, 20 minutes) or PSC IgG BEC-Ab negative (0.5 mg/mL, 20 minutes) or TNF-α (20 ng/mL, 10 minutes). Cell signaling pathway was studied using cell ELISA (see Materials and Methods for details). For the Western blots, cell lysates were collected and immunoblotted with Ab against endogenous nuclear factor κB, ERK1/2, p38, and stress-activated protein kinase/c-Jun-N-terminal kinase and their respective phosphorylated Ab. β-actin was probed as the loading control. Both ELISA and Western blots showed that PSC IgG selectively phosphorylated ERK1/2 and nuclear factor κB. Results are means ± SD (n = 3). p-, phospho-.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
(A) Human BECs were either left untreated or preincubated with PSC IgG (0.5 mg/mL, 20 minutes) or normal IgG (0.5 mg/mL, 20 minutes) or TNF-α (20 ng/mL, 10 minutes). Cell lysates were collected and immunoblotted with Ab against endogenous and phosphorylated ELK-1. β-actin was probed as the loading control. PSC IgG phosphorylated ELK-1, the downstream substrate of ERK 1/2. (B) The ERK inhibitor PD98059 at various concentrations (as indicated) significantly inhibited phosphorylation of ELK-1. (C) Phosphorylation of ELK-1 was quantitated by densitometric analysis using Imager (Quantity one, version 1.2) and normalized to control levels (PSC IgG–treated cells without the inhibitor) arbitrarily set to 100%. Results are means ± SD (n = 3). *P < .05, **P < .001 compared with nontreated cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
A representative picture (n = 3) of flow cytometric analysis demonstrating no binding of normal IgG to human BECs or any effect of inhibition with the ERK specific inhibitor PD98059 on BECs. On the other hand, PSC IgG induced expression of both TLR4 and TLR9 on BECs (gray lines). However, various concentrations (as indicated) of PD98059 significantly abrogated the expression of TLR4 but not TLR9 on BECs in a dose-dependent manner. Secondary Ab were used as negative control (black lines).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
Immunohistochemical detection of TLR expression in liver biopsy sections. (A) Control staining with only the secondary Ab. (B–D) No TLR4 or TLR9 expression in bile ducts of PBC, autoimmune hepatitis, or normal livers was observed. (E and F) However, expression of TLR4 in bile ducts (black/brown staining, arrows) was observed in livers of patients with PSC with BEC-Ab. Positive staining of Kupffer cells was also observed (arrowhead). Results from 2 different patients are shown. (G and H) Similarly, expression of TLR9 (black staining) on bile ducts was also observed in patients with PSC with BEC-Ab. Results from 2 different patients are shown. The sections were counterstained with hematoxylin. (Original magnification: A–H, 40×.)
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

8. Figure 7
Immunohistochemical and immunofluorescence detection of Ig deposition and TLR expression in PSC liver biopsy sections. (A) Results from 4 different patients with PSC showing binding of anti-human IgG Ab to bile ducts in the liver sections of patients with PSC are shown. (B) Double staining of PSC liver sections with only secondary Ab, anti-human IgG (CY-3, red) and anti-TLR4 (FITC, green) or anti-human IgG (CY-3, red) and TLR9 (FITC, green), clearly showed coincident sites of reactivity (orange/yellow) in many areas.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions