1. Crohn’s disease and the NOD2 gene: a role for paneth cells
Sanjay Lala, Yasunori Ogura, Caroline Osborne, Sok Ying Hor, Annabel Bromfield, Susan Davies, Olagunju Ogunbiyi, Gabriel Nuñez, Satish Keshav 
Gastroenterology 
Volume 125, Issue 1, Pages (July 2003)
DOI: /S (03)

2. Figure 1
NOD2 RNA is expressed by Paneth cells in small intestinal crypts. (A ) In situ hybridization with the digoxigenin-labeled NOD2 antisense strand riboprobe shows positive brown staining in small intestinal crypts (arrowheads) in CD-affected terminal ileum. (B) Hybridization with the control sense strand riboprobe shows no signal, confirming specificity of detection. (C ) Within crypts, positive staining is present mainly in the infranuclear region of Paneth cells (thick arrows), where RNA and rough endoplasmic reticulum is localized, whereas the protein-rich granules in the apical cytoplasm (asterisks) are unstained. Positive signal also is noted in lamina propria mononuclear cells (thin arrows). (D) Sections hybridized with the control sense strand riboprobe show no positive staining. (E ) NOD2-positive crypt cells are identified as Paneth cells by localizing lysozyme RNA in adjacent sections hybridized with the lysozyme antisense strand riboprobe. (F ) Sections hybridized with the control sense strand riboprobe show no staining. All sections were counterstained with hematoxylin, and viewed under (A, B) the ×20 objective, or (C, D) the ×100 objective.
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3. Figure 2
NOD2 protein is expressed by Paneth cells in small intestinal crypts. (A ) In CD-affected terminal ileum, positive immunohistochemical staining, using a purified rabbit antibody raised against recombinant NOD2 protein, is seen in the basal and apical cytoplasm in Paneth cells (thick arrow) and in a monocyte in the lamina propria (thin arrow). (B-D) Positive immunohistochemical staining of a weaker intensity is seen in Paneth cells (∗) in histologically normal (B) terminal ileal, (C ) jejunal, and (D) duodenal tissue. (E ) No staining is seen in CD-affected terminal ileal sections incubated with an unrelated rabbit antibody. (F ) No staining is seen in CD-affected terminal ileal sections incubated without primary antibody. All sections were counterstained with hematoxylin, and viewed under the ×100 objective.
Gastroenterology  , 47-57DOI: ( /S (03) )

4. Figure 3
NOD2 RNA is expressed by metaplastic Paneth cells in CD-affected colonic mucosa. (A ) In situ hybridization with the digoxigenin-labeled NOD2 antisense strand riboprobe shows positive brown staining in metaplastic Paneth cells (thick arrows) present in the crypt epithelium in CD-affected colon, and a positive signal also is seen in lamina propria mononuclear cells (thin arrows). (B) Sections hybridized with the control sense strand riboprobe show no staining. (C ) NOD2-positive colonic epithelial cells are identified as metaplastic Paneth cells (thick arrows) by localizing lysozyme RNA in adjacent sections hybridized with the lysozyme antisense strand riboprobe. (D) Sections hybridized with the control sense strand riboprobe show no staining. All sections were counterstained with hematoxylin, and viewed under the ×40 objective.
Gastroenterology  , 47-57DOI: ( /S (03) )

5. Figure 4
Expression and quantification of NOD2 mRNA in microdissected Paneth cells and crypt epithelial cells. (A ) Sections of small intestine before and after laser-capture microdissection of Paneth cells and villus epithelial cells are shown stained with hematoxylin-eosin and viewed under the ×10 objective. The upper panels show Paneth cells and the lower panels show villus epithelial cells, before microdissection (left-most panels) and after microdissection (middle panels). Microdissected cells captured on membranes are shown in the right-most panels. (B) NOD2, defensin 6, and GAPDH mRNA were analyzed by nested RT-PCR using intron-spanning primer pairs, in samples of LCM-acquired Paneth cells and villus epithelial cells. For samples with equivalent GAPDH expression, NOD2 mRNA expression is detected readily in Paneth cells but not in villus epithelial cells. Defensin 6 is detected in both Paneth cells and villus epithelial cells, with much lower levels in the villus epithelial fraction. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (C ) Quantitative real-time RT-PCR analysis of crypt and villus epithelium shows enrichment of NOD2 and defensin 6 mRNA expression in crypts compared with villi. For comparison, NOD2 expression in freshly isolated PBMCs also is shown, and this is approximately 2.7-fold greater than in crypts. The relative expression of NOD2, defensin 6, and GAPDH mRNA was determined by real-time RT-PCR, using intron-spanning primers, and the NOD2:GAPDH and defensin 6:GAPDH ratios calculated for each sample. Results are shown as the relative expression compared with the level in crypts.
Gastroenterology  , 47-57DOI: ( /S (03) )

6. Figure 5
NOD2 RNA is expressed by monocytes in inflamed intestinal tissue. (A ) In situ hybridization with the digoxigenin-labeled NOD2 antisense strand riboprobe shows NOD2 RNA in newly recruited monocytes (arrows) located on the periphery of a granuloma, and not in centrally located histiocytes and epitheloid macrophages (∗) in CD-affected terminal ileum. (B) Hybridization with the control sense strand riboprobe shows no signal, confirming specificity of detection. Asterisk indicates the central region of the granuloma. (C ) Immunohistochemical staining using an anti-CD68 antibody positively identifies newly recruited monocytes (arrows) located on the periphery of a granuloma, and mature macrophages (open arrows) located centrally within a granuloma. (D) In situ hybridization with the NOD2 antisense strand riboprobe shows positive signals in lamina propria monocytes (arrows), which are identified by their nuclear morphology, but not in other leukocytes. (E ) No positive signal is detected in sections hybridized with the control sense strand riboprobe. (F ) Immunohistochemical staining using an anti-CD68 antibody verifies the identity of lamina propria monocytes (arrows). All sections were counterstained with hematoxylin, and viewed under (A-C the ×40 objective, or (D-F the ×100 objective.
Gastroenterology  , 47-57DOI: ( /S (03) )

7. Figure 6
NOD2 mRNA expression in PBMCs and intestinal epithelial cells. (A ) Expression of NOD2 mRNA was quantified in freshly isolated PBMCs; PBMCs maintained in culture for 24, 48, and 72 hours; and in Caco-2 and HT29 cells, using real-time RT-PCR with intron-spanning primers. Results were normalized using GAPDH mRNA as an internal standard. NOD2 mRNA levels are shown as a percent relative to the level in freshly isolated PBMCs. (B) RT-PCR using intron-spanning primers showed NOD2 and GAPDH amplification products from mRNA extracted from freshly isolated PBMCs; PBMCs maintained in culture for 24, 48, and 72 hours; and Caco-2 and HT29 cells, corresponding to A. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining.
Gastroenterology  , 47-57DOI: ( /S (03) )

8. Figure 7
TNFα RNA is strongly expressed in Paneth cells in Crohn’s ileitis. (A ) In situ hybridization with the digoxigenin-labeled TNFα antisense strand riboprobe shows no positive staining in Paneth cells in histologically normal terminal ileum. (B) Parallel sections hybridized with the control sense strand riboprobe show no positive staining. (C ) In situ hybridization with the TNFα antisense strand riboprobe shows positive staining in Paneth cells (arrow) in CD-affected terminal ileum. (D) Hybridization with the control sense strand riboprobe shows no signal, confirming the specificity of detection. (E ) In situ hybridization with the TNFα antisense strand riboprobe shows positive signal in lamina propria monocytes in CD-affected terminal ileum. (F ) No positive signal is detected in sections hybridized with the control sense strand riboprobe. All sections were counterstained with hematoxylin, and viewed under the ×100 objective.
Gastroenterology  , 47-57DOI: ( /S (03) )