Card15 gene overexpression in mononuclear and epithelial cells of the inflamed Crohn's disease colon

Abstract

Background: Crohn's disease is one of the principal human chronic inflammatory bowel diseases. Although its aetiology is still unknown, its complex pathogenesis has environmental, immunological, and genetic determinants. CARD15 is the first susceptibility gene implicated in the predisposition to Crohn's disease and is known to be expressed only in monocytes. However, its expression in situ has not yet been studied.

Aims: To analyse the tissue distribution of CARD15 and identify cells producing CARD15 in samples of colon from patients with Crohn's disease and control subjects.

Patients and methods: We analysed CARD15 gene expression in surgical specimens of colon from eight children with Crohn's disease and nine controls by immunohistochemistry, in situ hybridisation, and reverse transcription-polymerase chain reaction (RT-PCR).

Results: We showed that CARD15 was present only in the cytoplasm of macrophages in the normal colon. Increased CARD15 expression was detected in Crohn's disease lesions. There were more CARD15 positive cells in Crohn's disease lesions than in uninvolved areas. Both intestinal epithelial cells, macrophages, and their derivatives overproduced CARD15 in Crohn's disease. To further assess CARD15 expression by intestinal epithelial cells, we performed RT-PCR on freshly isolated intestinal epithelial cells, and showed that these cells isolated from Crohn's disease samples contained more CARD15 mRNA than intestinal epithelial cells from controls.

Conclusions: We have demonstrated that colonic involvement in active Crohn's disease is associated with increased CARD15 gene expression in both macrophages and intestinal epithelial cells. Therefore, this deregulation can affect the host-environment interaction and thus contribute to the pathogenesis of this disease.

Figure 1

Immunohistochemistry using an anti-CARD15 polyclonal antibody and frozen tissue sections of normal colon (A) and inflamed Crohn’s disease colon (B–E), and subcellular distribution of CARD15 in human cells by confocal fluorescence microscopy (F). (A) Scattered mononuclear cells just beneath the epithelium surface, which look like macrophages (insert), are present in the normal mucosa. The Crohn’s disease colon contains numerous positive cells in the lamina propria whereas no CARD15 expression was detected within the lymphoid follicle (B, insert (*)). Positive cells were also detected in the deeper part of the colon and around the blood vessels (C, insert). Epithelioid and giant cells in a granuloma (D) and superficial and glandular epithelial cells also produced CARD15 (B, insert; C, E). (E, inserts) Negative controls (using recombinant protein and primary antibody omission) showed no background staining. (F) Nuclei were stained with DAPI (blue). Original magnification ×160 in (B), ×250 in (A), ×400 in (A, insert), (C), (D), and (E, insert), and ×1000 in (E).

Figure 2

In situ hybridisation using a CARD15 antisense probe (A–D) and frozen tissues sections of normal colon (A) and inflamed Crohn’s disease colon (B–D). (A) Only mononuclear cells contained CARD15 mRNA, and no CARD15 mRNA was detected in epithelial cells. Many cells expressing CARD15 mRNA were present in the CD mucosa (B). Epithelial cells expressing CARD15 mRNA (C) were identified by double labelling, detection of CARD15 by in situ hybridisation, and epithelial cells by immunohistochemistry using an anticytokeratin antibody (D). The sense probe gave no signal (E). Original magnification ×250 in (B), ×1000 in (A), (C), (D), and (E).

Figure 3

Immunohistochemistry using an anti-CARD15 polyclonal antibody (A) and in situ hybridisation using a CARD15 antisense probe (B) and frozen tissue sections from acute appendicitis cases. The inflamed appendix contains numerous positive cells in the lamina propria and in the submucosa whereas no CARD15 expression was detected within the lymphoid follicle (B). Superficial and glandular epithelial cells also produced CARD15. Original magnification ×250 in (A), and ×160 in (B).

Figure 4

Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CARD15 in whole colon tissues samples (A) and in colon epithelial cells of controls (C) and Crohn’s disease (CD) patients (B, C). (A) Ratio of CARD15 to β-actin mRNAs levels demonstrated a significant increase in whole tissue samples from CD patients compared with tissues samples from controls. Each bar represents the mean (SEM). Amplified products were subjected to gel electrophoresis and analysed by computed assisted densitometry with NIH Image 1.62 software. (B) CARD15 was detected in all intestinal epithelial cells extracted from surgical specimens of all patients with CD and controls enrolled in this study. (C) The level of CARD15 mRNA was more than five times higher in extracted epithelial cells from CD patients compared with controls. For equivalent quantities of β-actin, similar levels of CARD15 mRNA were found in extracted normal epithelial cells compared with peripheral blood mononuclear cells (PBMC). The number of patients (n) is indicated. OD, optical density.