Okada Kensaku, Chikumi Hiroki, Takata Miyako, Yamaguchi Kosuke, Makino Haruhiko, Kitaura Tsuyoshi, Nakamoto Masaki, Yamasaki Akira, Igishi Tadashi, Burioka Naoto, Shimizu Eiji
Division of Medical Oncology and Molecular Respirology, Department of Multidisciplinary Internal Medicine, School of Medicine,Tottori University Faculty of Medicine, Yonago 683-8504, Japan.
Division of Medical Oncology and Molecular Respirology, Department of Multidisciplinary Internal Medicine, School of Medicine,Tottori University Faculty of Medicine, Yonago 683-8504, Japan ; †Center for Infectious diseases, Tottori University Hospital, Yonago 683-8504, Japan.
Yonago Acta Med. 2015 Mar;58(1):31-8. Epub 2015 Mar 27.
Clarithromycin is a macrolide antibiotic that possesses anti-inflammatory and immunomodulatory properties. Although recent data suggests that macrolide antibiotics enhance Pseudomonas aeruginosa clearance from the lung, involving natural killer (NK) T cells in this process by activating the NKG2D-NKG2D ligand system, the precise underlying mechanism is still unclear. In this study, we examined the effect of clarithromycin on a potent NKG2D ligand, UL16-binding protein 2 (ULBP2), in the lung and its shedding mechanism.
The gene expressions of ULBP2 and the shredder proteinases of ULBP2, a disintegrin and metalloproteinase domain 10 (ADAM10) and ADAM17, were measured using real-time PCR. The cell surface ULBP2 expression was measured by flow cytometry. The amount of solubilized ULBP2 (sULBP2) was measured using an ELISA. The activity of ADAM17 was examined by measurement of fluorescence intensity from the fluorescence resonance energy transfer peptide substrate cleaved by ADAM17.
Clarithromycin significantly induced transcription of ULBP2 and ADAM17 in both A549 and LCSC #2 cells, which endogenously express minimal and abundant levels of ULBP2, respectively. However, there was no significant change on transcription of ADAM10. The same tendency was observed when LCSC #2 cells were treated with tumor necrosis factoralpha processing inhibitor-2 to inhibit ADAM17 activity. The amount of sULBP2 was significantly decreased in both A549 and LCSC #2 cells by treatment with clarithromycin. Finally, clarithromycin significantly inhibited the activity of ADAM17 in LCSC #2 cells.
These findings suggest that clarithromycin induces ULBP2 expression and reduces the amount of sULBP2, by possibly inhibiting the activity of the potent ULBP2-shedding enzyme ADAM17. Because these changes in ULBP2 and sULBP2 levels could activate NKT cells, this finding might indicate a novel mechanism by which clarithromycin improves the clearance of P. aeruginosa in chronic respiratory diseases.
克拉霉素是一种具有抗炎和免疫调节特性的大环内酯类抗生素。尽管最近的数据表明大环内酯类抗生素可增强铜绿假单胞菌从肺部的清除，在此过程中通过激活NKG2D-NKG2D配体系统涉及自然杀伤（NK）T细胞，但确切的潜在机制仍不清楚。在本研究中，我们研究了克拉霉素对肺部一种强效NKG2D配体UL16结合蛋白2（ULBP2）的影响及其脱落机制。
使用实时PCR测量ULBP2及其切割蛋白酶（一种去整合素和金属蛋白酶结构域10，即ADAM10）和ADAM17的基因表达。通过流式细胞术测量细胞表面ULBP2的表达。使用酶联免疫吸附测定法测量可溶性ULBP2（sULBP2）的量。通过测量ADAM17切割的荧光共振能量转移肽底物的荧光强度来检测ADAM17的活性。
克拉霉素显著诱导A549和LCSC #²细胞中ULBP2和ADAM17的转录，这两种细胞分别内源性表达极低和丰富水平的ULBP2。然而，ADAM10的转录没有显著变化。当用肿瘤坏死因子α加工抑制剂-2处理LCSC #²细胞以抑制ADAM17活性时，观察到相同的趋势。用克拉霉素处理后，A549和LCSC #²细胞中sULBP2的量均显著降低。最后，克拉霉素显著抑制LCSC #²细胞中ADAM17的活性。
这些发现表明，克拉霉素可能通过抑制强效的ULBP2脱落酶ADAM17的活性来诱导ULBP2表达并减少sULBP2的量。由于ULBP2和sULBP2水平的这些变化可能激活NKT细胞，这一发现可能表明克拉霉素改善慢性呼吸道疾病中铜绿假单胞菌清除的一种新机制。
