Keratinocyte growth factor up-regulates Interleukin-7 expression following intestinal ischemia/reperfusion in vitro and in vivo

Abstract

Background: Epithelial cell (EC)-derived Interleukin-7 (IL-7) plays a crucial role in control of neighboring intestinal intraepithelial lymphocytes (IEL) development and homeostasis, and IEL derived keratinocyte growth factor (KGF) promotes intestinal epithelial growth, which was regulated by EC-derived IL-7. On this basis, we hypothesize that there is a crosstalk between IELs and ECs, and KGF could regulate the EC-derived IL-7 expression, which should be associated with the protective effects by KGF on intestinal injury.

Methods: Histological evaluation was performed in small intestine tissues of patients with intestinal obstruction and IL-7 expression was detected by immunofluorescence. Intestinal epithelial cells (LoVo) and adult C57BL/6J mice undergoing ischemia/reperfusion injury were treated with recombinant KGF. KGF, KGF receptor(KGFR) and IL-7 expressions were measured with western blot and immunofluorescence analysis.

Results: IL-7 expression increased in the mild ischemia while decreased in severe ischemia small intestinal tissues of patients with intestinal obstruction. KGF expression significantly decreased while IL-7 expression increased early after acute intestinal I/R administration in a mouse model. KGF treatment significantly increased the IL-7 expression both in vitro and in vivo, while when the KGFR was blocked, the findings above were absent. In addition, our results showed changes of IL-7 expression at different stages after acute intestinal I/R administration, KGF treatment significantly attenuated the decreasing of IL-7 expression caused by acute intestinal I/R.

Conclusion: KGF could up-regulate the IL-7 expression both in vitro and in vivo through KGFR pathway, which should have associated with the protective effects of KGF in intestinal injury.

Keywords: Keratinocyte growth factor; epithelial cells; interleukin-7; ischemia/reperfusion; mouse.

Figure 1

Histology changes in small intestine tissues of patients. More severe mucosal injury was observed in severe ischemia group (A) than mild ischemia group (B), while none in normal tissues (C); Original magnification × 200.

Figure 2

Alterations in IL-7 immunofluorenscence staining of small intestine tissues of patients. IL-7 expression was significantly decreased in severe ischemia small intestinal tissues (A), while increased in mild ischemia small intestinal tissues (B), as compared with normal tissues (C); (D) Arithmetic means ± SEM of IOD of IL-7 immunofluorenscence staining in small intestinal tissues of patients, * P < 0.05, between ischemia injury group and normal.

Figure 3

Changes of IL-7 and KGF expression in a mouse model of acute intestinal I/R.(A) Decreased expression of KGF and increased expression of IL-7 were confirmed by western blot in mice early after acute intestinal I/R, compared with sham. Tublin was used as an internal control. (B) Spearman rank correlation of KGF and IL-7 protein levels with acute intestinal I/R in mice. *indicates significant difference between I/R administration group and sham (P < 0.05), n = 6 per group.

Figure 4

KGF administration regulates IL-7 and KGFR expression both in vivo and in vitro.(A)Increased expression of IL-7 and decreased expression of KGFR were confirmed by western blot in LoVo cells with KGF (40ng/ml) treatment. Tublin was used as internal control. * P < 0.05, between KGF treated group and control. (B) Increased expression of IL-7 and decreased expression of KGFR were confirmed by western blot in mice with KGF (5 mg/kg/ day×5d) treatment. Tublin was used as an internal control. *indicates significant difference between KGF treated group and control (P < 0.05).

Figure 5

KGF administration results in an increased IL-7 expression both in vivo and in vitro.(A) Dose-dependent increased expression of IL-7 was confirmed by western blot in LoVo cells with KGF treatment. Tublin was used as an internal control. IL-7 expression increased nearly 4-folds when the concentration of KGF was at 150 ng/ml. *indicates significant difference between KGF treated group and control (P < 0.05). (B) Increased expression of IL-7 was confirmed by immunofluorenscence staining in mice with KGF treatment. * P < 0.05, between KGF treated group and control.

Figure 6

Changes of IL-7 expression in response to KGF treatment in a mouse model of ischemia-reperfusion injury. (A) Changes of IL-7 expression were confirmed by western blot in acute intestinal I/R mice with KGF (5 mg/kg/day×5d) treatment. Increased IL-7 expression was highest at 6h after I/R administration with KGF treatment, while decreased the lowest at 24h after I/R administration without treatment. * P < 0.05, between I/R administration group and sham, ** P < 0.05, between KGF treated group and untreated (control).(B)Confocal laser scanning microscopic analysis of IL-7 in mice intestinal paraffin sections stained with immunofluorenscence, showing specific FITC-related fluorescence in the cytoplasm. Visualization of the nuclei was evident by DAPI staining. At different time points (0, 6h, 24h and 48h after intestinal I/R administration), changes of IL-7 expression in mice were observed, with or without KGF treatment.

Figure 7

IL-7 is up-regulated by KGF through KGFR pathway. Tublin was used as an internal control. (A)Decreased expression of IL-7 was confirmed by western blot in LoVo cells after KGFR blockade with KGF treatment. Suppression of IL-7 expression was observed with dose dependent of KGFR antibody blockage (5μg/μl and10μg/μl) following KGF (50ng/ml and 100ng/ml) treatment. * P < 0.05, between KGFR blockade following KGF treated group and KGF (50ng/ml) treated group. (B) Reduced expression of KGFR was confirmed by western blot in LoVo cells following KGFR RNA interference. Plasmids 335, 336 and 337 were transfected into LoVo cells and KGFR expression was detected. Plasmid 335 and 337 can definitely inhibit the KGFR expression. Expressions of Tublin and KGFR in LoVo cells transfected with plasmid 336 were both very low, which suggested the plasmid 336 treated cells were unqualified for experiment. * P < 0.05, between KGFR RNA interference group and plasmid control. (C)Reduced expression of IL-7 was confirmed by western blot in LoVo cells following KGFR RNA interference. Decreased expressions of IL-7 were observed in LoVo cells following KGF treatment in response to RNA interference of KGFR by plasmid 335, plasmid 337 and plasmid 335+337. * P < 0.05, between plasmid+KGF group and control+KGF.