Volume 122, Issue 7, Pages 1987-2000 (June 2002) Toll-like receptors 2 and 4 are up-regulated during intestinal inflammation  M. Hausmann, S. Kiessling, S. Mestermann, G. Webb, T. Spöttl, T. Andus, J. Schölmerich, H. Herfarth, K. Ray, W. Falk, G. Rogler  Gastroenterology  Volume 122, Issue 7, Pages 1987-2000 (June 2002) DOI: 10.1053/gast.2002.33662 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Western blot to characterize the specificity of the newly generated antibodies. (A) Western blot analysis with recombinant proteins (extracellular domain) revealed a single band at the expected size for both anti-TLR2 and -TLR4 antibodies. (B) Western blot analysis with anti-TLR2 antibody revealed no signal for mock or TLR1-transfected cells (THP-1) as indicated, but a single band for anti-TLR2–transfected cells (left). Analysis with anti-TLR4 antibody revealed a double band for endogenous TLR4 in THP-1 (right), which was further increased after stimulation with LPS or transfection with TLR4. No signal was obtained with pre-absorbed peptide. Gastroenterology 2002 122, 1987-2000DOI: (10.1053/gast.2002.33662) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Frozen sections were cut and fixed in acetone for peroxidase staining. Immunohistochemical detection of TLR2 in control mucosa and inflamed mucosa. (A) No cells containing TLR2 were detectable in control mucosa from non-inflamed patients. (B) In CD, cells with TLR2 were detectable in the lamina propria. (C) In UC, cells with TLR2 were detectable in the lamina propria. (D) Detection of TLR2 in SD to a lesser degree (original magnification 400×). Gastroenterology 2002 122, 1987-2000DOI: (10.1053/gast.2002.33662) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Frozen sections were cut and fixed in acetone for peroxidase staining. Immunohistochemical detection of TLR4 in the inflamed mucosa. (A) No cells containing TLR4 were detectable in control mucosa from non-inflamed patients. (B) In CD, cells with TLR4 were detectable in the lamina propria. (C) In UC, cells with TLR4 were detectable in the lamina propria. (D) Detection of TLR4 in SD to a lesser degree (original magnification 400×). Gastroenterology 2002 122, 1987-2000DOI: (10.1053/gast.2002.33662) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Immunofluorescent and immunohistochemical detection of TLR2 and TLR4 co-expression in the inflamed mucosa. TLR2 was visualized by rabbit anti-human antibody, and TLR4-positive cells were detectable with mouse anti-human antibody. TLR2 was stained in a second step with Alexa Fluor 488 conjugated goat anti-rabbit antibody (green fluorescent, A and B, upper) or NovaRED (red) reaction product (C and D, upper). TLR4 was stained in a second step with Alexa Fluor 546 conjugated goat anti-mouse antibody (red fluorescent, lower). TLR2 could be co-localized in most of the TLR4-positive cells and vice versa (original magnification 400×). Gastroenterology 2002 122, 1987-2000DOI: (10.1053/gast.2002.33662) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Identification of the cell types containing TLR2 in the inflamed mucosa. Cellular markers were stained in a first step by anti-CD68, anti-CD14, or anti-CD66b antibody. TLR2 was stained in a second step. Positive cells were visualized with NovaRED (red) and BDHC (dark blue) reaction product. (A) No TLR2 was detected in macrophages (blue) from non-inflamed mucosa. TLR2 (red) could be co-localized in macrophages (blue) from (B) a CD patient and (C) a UC patient. Applying the staining reagents in reverse order, TLR2 (blue) could be co-localized in macrophages (red) from (D) a CD patient, (E) a UC patient, and (F) a patient with SD. (G) TLR2 (blue) could be co-localized in CD14-positive cells (red) from a UC patient. TLR2 (blue) could not be co-localized in neutrophils (red) from (H) a CD patient and (I) a UC patient. (J) No BDHC reaction product was detected using rabbit control serum in the second staining step. (K) No NovaRED reaction product was detected using mouse isotype control in the first staining step (original magnification 400×, except G, 100×). Gastroenterology 2002 122, 1987-2000DOI: (10.1053/gast.2002.33662) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Identification of the cell types containing TLR4 in the inflamed mucosa. (A) No TLR4 was detected in macrophages (blue) from non-inflamed mucosa. TLR4 (red) could be co-localized in macrophages (blue) from (B) a CD patient and (C) a UC patient. Applying the staining reagents in reverse order, TLR4 (blue) could be co-localized in macrophages (red) from (D) a CD patient, (E) a UC patient, and (F) a patient with SD. (G) TLR4 (blue) could be co-localized in CD14-positive cells (red) from a UC patient. TLR4 (blue) could not be co-localized in neutrophils (red) from (H) a CD patient and (I) a UC patient (original magnification 400×, except G, 100×). Gastroenterology 2002 122, 1987-2000DOI: (10.1053/gast.2002.33662) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 TLR mRNA expression in freshly elutriated monocytes, intestinal macrophages from a patient with non-inflamed mucosa and a CD patient. (A) RT-PCR for TLR1–5 in monocytes as indicated. RT-PCR for TLR1–5 and β-actin (right) in CD33-positive cells (B) from a patient without intestinal inflammation and (C) from a CD patient. Monocytes express TLR1–5 mRNA. Only intestinal macrophages from mucosa of CD patients express significant amounts of TLR2, TLR4, and TLR5 mRNA. This Figure is representative for 2 more experiments. Gastroenterology 2002 122, 1987-2000DOI: (10.1053/gast.2002.33662) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 So-called virtual Northern blot analysis with intestinal macrophages cDNA from CD patients and macrophages from non-inflamed (NI) patients. Virtual Northern blot for TLR2, TLR4, and β-actin (lower lane) with macrophages isolated from 4 different patients each. To avoid individual differences responsible for virtual Northern blot results, macrophages from 4 inflamed specimens and from 4 normal specimens were pooled. Only macrophages from mucosa of CD patients express significant amounts of TLR2 mRNA. This blot is representative for 2 more experiments. Gastroenterology 2002 122, 1987-2000DOI: (10.1053/gast.2002.33662) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 Flow cytometry analysis of TLR2 expression in human elutriated monocytes and intestinal macrophages. Cells were stained with anti-TLR2 antibody (gray histogram) or control (white histogram). (A) TLR2 expression in elutriated monocytes. (B) TLR2 expression in macrophages in mucosa without inflammation. (C) TLR2 expression in macrophages in UC. Only elutriated monocytes and macrophages from mucosa of UC patients showed expression of TLR2. Gastroenterology 2002 122, 1987-2000DOI: (10.1053/gast.2002.33662) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 10 Flow cytometry analysis of TLR4 expression in human elutriated monocytes and intestinal macrophages. Cells were stained with anti-TLR4 antibody (gray histogram) or control (white histogram). (A) TLR4 expression in elutriated monocytes. (B) TLR4 expression in macrophages in mucosa without inflammation. (C) TLR4 expression in macrophages in UC. Only elutriated monocytes and macrophages from mucosa of UC patients showed expression of TLR4. Gastroenterology 2002 122, 1987-2000DOI: (10.1053/gast.2002.33662) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 11 Quantification of human IL-1–mRNA. IL-1–mRNA production of monocytes (left), in vitro differentiated macrophages (middle), and intestinal macrophages from non-inflamed mucosa (right; Quantitative PCR Detection Kit [CytoXpress, Biosource]). Cells were either not stimulated (white columns) or stimulated with 1 ng/mL (light gray columns), 10 ng/mL (dark gray columns), and 100 ng/mL (black columns) LPS. CD33-positive cells from non-inflamed intestinal mucosa fail to express IL-1—mRNA. Gastroenterology 2002 122, 1987-2000DOI: (10.1053/gast.2002.33662) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 12 Flow cytometry analysis of human intracellular IL-1 protein in LPMCs. IL-1 protein production of LPMCs from non-inflamed intestinal mucosa (NI), and from inflamed intestinal mucosa from CD patients with low macroscopic (endoscopical; L-CD) and high degree of inflammation (H-CD). LPMCs were isolated and stimulated for 12 hours with LPS (10 ng/mL). The percentage of cells positive for IL-1 protein was determined by flow cytometry. Only LPMCs from intestinal mucosa with high degree of macroscopic inflammation showed expression of IL-1 protein. Gastroenterology 2002 122, 1987-2000DOI: (10.1053/gast.2002.33662) Copyright © 2002 American Gastroenterological Association Terms and Conditions