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. 2000 Aug;47(2):192–198. doi: 10.1136/gut.47.2.192

Cytokine gene expression during postnatal small intestinal development: regulation by glucocorticoids

C Schaeffer 1, M Diab-Assef 1, M Plateroti 1, F Laurent-Huck 1, J Reimund 1, M Kedinger 1, C Foltzer-Jourdainn 1
PMCID: PMC1728015  PMID: 10896909

Abstract

BACKGROUND—In the intestinal mucosa, numerous cytokines produced by the epithelium, fibroblasts, and immune cells were shown to affect epithelial differentiation and proliferation through epithelial-mesenchymal and epithelial-immune cell interactions. To date, the importance of cytokines in postnatal development of the rat small intestine has not been established.
AIM—To investigate the developmental changes in expression of mucosal cytokines in the postnatal maturation of the rat small intestinal epithelium and their regulation by glucocorticoids (GC).
METHODS—Mucosal maturation was assessed by the onset of sucrase-isomaltase (SI) mRNA, analysed by in situ hybridisation. The amount of transforming growth factor β1 (TGF-β1), β2 (TGF-β2), tumour necrosis factor α (TNF-α), interleukin 1β (IL-1β), and TGF-α was analysed by reverse transcription-polymerase chain reaction (RT-PCR) in mucosal extracts from weaning (14-21 days old) and adult rats, or one day after an injection of hydrocortisone (HC) in 11 day old rats. Similarly, expression of cytokines and the regulatory effect of GC were studied on cultured subepithelial myofibroblasts cloned from postnatal jejunum and ileum cultured in the absence or presence of dexamethasone (DX).
RESULTS—TGF-β1, TGF-β2, and IL-1β decreased during the third week of life while levels of TNF-α increased and TGF-α remained constant. In parallel, SI transcripts increased and showed a progressive accumulation in the apical part of the enterocytes first localised at the base of the villi from 18 days onwards. Interestingly, precocious induction of SI mRNA by HC paralleled the decrease in expression of TGF-β isoforms and of IL-1β. All cytokines were expressed in the myofibroblast cell lines. In addition, the results showed that TNF-α was differentially expressed in basal culture conditions and after DX stimulation in jejunal and ileal myofibroblasts. DX decreased IL-1β but not the TGF-β isoforms, similar to that in vivo.
CONCLUSIONS—This study shows that mucosal cytokines are developmentally regulated and that GC are potentially involved in this regulation in parallel with maturation of the gut mucosa at weaning.


Keywords: small intestine; weaning; maturation; fibroblasts; cytokines

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Figure 1  .

Figure 1  

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Representative illustration of the developmental pattern of cytokine expression in the small intestinal mucosa from suckling rats. (A) RT-PCR analysis of transforming growth factor β1 and β2 (TGF-β1, TGF-β2), transforming growth factor α (TGF-α), tumour necrosis factor α (TNF-α), and interleukin 1β (IL-1β) in the duodenum, proximal jejunum, and distal ileum of 14, 18, 21, and 60 day old rats. (B) Densitometric analysis of the specific bands normalised to the corresponding values of β-actin mRNA used as internal control; mean (SEM) of values (expressed as percentages of the relative intensity of the bands at 14 days) obtained from three independent experiments. Significant differences compared with values obtained in 14 day old suckling rats (unpaired t test): *p<0.05, **p<0.01.

Figure 2  .

Figure 2  

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Developmental pattern of sucrase-isomaltase (SI) mRNA expression. In situ hybridisation was performed on sections of the proximal jejunum using an SI antisense cRNA probe as described in materials and methods. Dark field (A, C, D) and bright field illuminations (B, E-H) of intestinal sections from 11 (A, B), 16 (C), 18 (D), 21 (E), and 60 (F-H) day old rats. Controls: hybridisation with a sense probe (G) or treatment of the section with RNAse before hybridisation (H). Magnifications: A, C-F ×50; B, G, H, ×250.

Figure 3  .

Figure 3  

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Effects of hydrocortisone (HC), injected in 11 day old pups, on sucrase-isomaltase (SI) induction and cytokine expression. (A) Representative RT-PCR assays detecting SI, transforming growth factor β1 and β2 (TGF-β1, TGF-β2), transforming growth factor α (TGF-α), tumour necrosis factor α (TNF-α), interleukin 1β (IL-1β), and β-actin mRNAs at postnatal day 12: lanes 1, control; lanes 2, treated animals. (B) Densitometric analysis of SI, TGF-β1, TGF-β2, TGF-α, TNF-α, and IL-1β bands normalised to values of β-actin as internal control from three independent experiments. Results are illustrated in the HC treated tissues as percentages of control values. *p< 0.05, ** p<0.01, significant differences between HC treated and control animals (unpaired t test).

Figure 4  .

Figure 4  

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Effect of DX on expression of cytokines by subepithelial myofibroblast cell lines. (A) Representative RT-PCR analysis of transforming growth factor β1 and β2 (TGF-β1, TGF-β2), transforming growth factor α (TGF-α), tumour necrosis factor α (TNF-α), interleukin 1β (IL-1β), and ribosomal protein transcripts extracted from myofibroblasts cloned from the jejunum (MIC 101-1; lanes 1 and 2) and ileum (MIC 216; lanes 3 and 4). Myofibroblastic cells were cultured for two days in the basic medium (lanes 1 and 3) and in the presence of 8×10 -7 M DX (lanes 2 and 4). (B) Densitometric analysis of TGF-β1, TGF-β2, TGF-α, IL-1β, and TNF-α bands (normalised to values of ribosomal protein used as internal control): mean of values obtained from the experiments performed on the two jejunal (MIC 101-1 and 101-2) or the two ileal (MIC 216 and 219) cell lines which gave identical results. Results are illustrated in the DX treated cultures as percentages of the corresponding controls. *p<0.05 compared with controls (unpaired t test).

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