FK506（タクロリムス）連続投与によるマウス小腸上皮細胞の形態的及び機能的変化

−UV励起アルカリ性フォスファターゼ基質ELF97 phosphateのflow cytometryへの応用−

抄録

We previously reported that anti-CD3 mAb treatment induced massive DNA fragmentation and the subsequent detachment of the mucosal epithelial cells of the intestine. To elucidate the role of intraepithelial lymphocytes (IELs) in the mucosal immunity of the small intestine, we designed experiments to inactivate the IEL by immunosuppressant, FK506 and to examine the subsequent changes of the enterocytes. Daily administration of FK506 for 2 weeks resulted in a decrease in both the potential cytotoxicity and the expression of TNF-" mRNA in i-IELs. The inactivation of i-IELs by daily administration of FK506 disappeared apoptosis of enterocytes in villus. Then, in the enterocytes, the long term daily adminisyration of FK506 caused decrease in the expression of amino acid transporter, CD98. Furthmore, these treatments induced the disappearance and heterogenous expression of F-actin in microvilli of enterocytes. Next, to estimate alkaline phosphatase (ALP) activity by flow cytometry (FCM), we established analysis of ALP activity using UV-excited fluorochrome,ELF97 phosphate and BD LSR flow cytometer. In same condition, comparison of FITC, PE or ALP plus ELP97 phosphate revealed that combination of ALP plus ELF97 phosphate showed the strongest intensity among these fluorochromes. Therefore, we used the combination of ALP and ELF97 phosphate to measure ALP activity by FCM. In the fixed enterocytes, the long term daily adminisyration of FK506 caused decrease in the ALP activity. These results were also comfirmed the histological examinations. Taken together, our study showed that the inactivation of i-IELs by daily administration of FK506 could inhibit apoptosis of enterocytes which resulted in the dysfunctional enterocytes remained in the villi.