Gene alteration of intestinal intraepithelial lymphocytes in response to massive small bowel resection
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The intestinal adaptive response [increased epithelial cell (EC) proliferation and apoptosis] after massive small bowel resection (SBR) is partially controlled by intraepithelial lymphocytes (IEL). To identify IEL factors contributing to EC adaptation post-SBR we utilized microarray assays.
Mice underwent a 70% SBR (SBR1w/SBR4w) or sham operation (Sham1w/Sham4w). After 1 or 4 weeks (1w, 4w) small bowel was harvested, and IEL isolated. Determination of the EC-proliferation rate used BrdU incorporation, and of the EC-apoptotic rate used Annexin V staining. Affymetrix system microarrays (12,491 genes) were performed to examine IEL-mRNA expression. Results were considered significant if fold-change (FC) between groups was >2 and P <0.05 (F-test), or FC>3 and 0.05> P >0.01, or FC>4 and P >0.05. Significant genes were confirmed by conventional RT-PCR.
The SBR EC-proliferation rate increased significantly in both 1w and 4w groups compared to Sham: SBR1w 0.24±0.07 vs. Sham1w 0.12±0.02 ( P =0.03); SBR4w 0.35±0.04 vs. Sham4w 0.19±0.02 ( P <0.01). The EC-apoptotic rate was unchanged in the 1w group, but significantly differed from controls after 4 weeks: SBR4w 39.92±6.78 vs. Sham4w 12.56±6.44 ( P <0.01). Microarray results were analyzed to identify potential growth-modifying IEL genes. The following were identified (function in parenthesis; A, apoptosis; P, proliferation): lipocalin 2 (promotes A), angiotensin converting enzyme (increases A), Rap2 interacting protein (reduces A, promotes P), amphiregulin (promotes P) and leucine-rich-α2-glycoprotein (promotes A, reduces P). Based on RT-PCR results these genes showed significant changes between groups. The increase in ACE at 1w preceded the observed apoptotic changes. The alterations in lipocalin 2, Rap2 and amphiregulin at 4w coincided with the marked changes in growth and apoptosis in the SBR mice.
IEL undergo temporal changes after SBR. These findings provide profound insight into potential IEL-dependent regulation of EC homeostasis post-SBR.
KeywordsShort bowel syndrome Intraepithelial lymphocytes Apoptosis Proliferation Microarray
This work was supported by a grant from the National Institute of Health, USA (AI44076–01), by fellowships from Novartis Stiftung Schweiz, Swiss National Foundation, and the Swiss Society of Pediatric Surgery. Microarray analysis was supported through the University of Michigan NIDDK Biotechnology Center.
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