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Effects of macrolides on proinflammatory epitops on endothelial cells in vitro

机译：大环内酯类药物对内皮细胞促炎表位的影响

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摘要

An inflammatory reaction at the site of infusion is a common clinical problem that is observed after the intravenous application of antibiotics and other drugs. The pathomechanism of this infusion-related phlebitis is not fully understood. We analyzed the effects of the three macrolide antibiotics erythromycin, clarithromycin and azithromycin on human endothelial cells in vitro. As a positive control quinupristin/dalfopristin was studied. The cytotoxicity of all substances was analyzed by a modified MTT cytotoxicity assay with 3T3-fibroblasts and EA.hy 926 endothelial cells. Cells were incubated for 10 days with the antibiotics. After adding MTT the optical density was measured which correlates with cell death. Clarithromycin exhibited the strongest cytotoxic effect on EA.hy 926 cells (EC50 30 mg/L), followed by azithromycin (EC50 40 mg/L), a cytotoxic effect of erythromycin could only be observed at much higher concentrations (EC50 310 mg/L). The reaction of the endothelial cells was further analyzed in detail by means of flow cytometry. For these experiments the endothelial cell line EA.hy 926 as well as primary cells (HUVEC) were used. The antigens were stained with fluoresceinisothiocyanat- or phycoerythrin-conjugated monoclonal antibodies for the following surface antigens: CD34, E-selectin (CD62E), ICAM-1 (CD54) and VCAM-1 (CD106). Cells were incubated with the antibiotics at concentrations ranging from 100 to 800 mg/L (clarithromycin and azithromycin) and from 200 to 1,200 mg/L (erythromycin). These concentrations occur under therapeutic conditions at the site of infusion. Cells were incubated for 2 h and analysis was carried out after an additional culture period of 22 h without test compounds. A significantly enhanced expression of all four antigens was observed which was most pronounced at 800 mg/L (erythromycin), 600 mg/L (azithromycin) and 400 mg/L (clarithromycin). A concentration of 800 mg/L erythromycin medium caused an increase of the expression of CD34 (+6%), E-selectin (+5%), ICAM-1 (+14%) and VCAM-1 (+5%). At lower concentrations (600 mg/L) azithromycin provokes a stronger upregulation of the proinflammatory antigens: CD34 (+17%), E-selectin (+18%), ICAM-1 (+27%) and VCAM-1 (+17%). At a concentration of 400 mg/L medium clarithromycin induced a similar effect as erythromycin at twice this concentration: CD34 (+5%), E-selectin (+7%), ICAM-1 (+23%) and VCAM-1 (+4%). Reactions of the HUVECs were less pronounced than those of the EA.hy 926 cells. Cell surface markers involved in interactions between endothelial cells and leukocytes proved to be useful markers to study differences in the proinflammatory potential of the three macrolides. By analysing the upregulation of these antigens on EA.hy 926 cells in vitro the risk of phlebitis could be predictable for other drugs as well.
机译：输注部位的炎症反应是常见的临床问题，在静脉内应用抗生素和其他药物后会观察到。这种与输液有关的静脉炎的发病机理尚未完全了解。我们分析了三种大环内酯类抗生素红霉素，克拉霉素和阿奇霉素对人内皮细胞的体外作用。作为阳性对照，研究了奎奴普丁/达福普汀。通过改良的MTT细胞毒性试验，使用3T3-成纤维细胞和EA.hy 926内皮细胞，分析了所有物质的细胞毒性。将细胞与抗生素一起温育10天。加入MTT后，测量与细胞死亡相关的光密度。克拉霉素对EA的细胞毒性最强。hy926细胞（EC 50 30 mg / L），其次是阿奇霉素（EC 50 40 mg / L），具有细胞毒性作用仅在高浓度（EC 50 310 mg / L）中才能观察到红霉素的含量。借助于流式细胞术进一步详细分析了内皮细胞的反应。对于这些实验，使用内皮细胞系EA.hy 926以及原代细胞（HUVEC）。用荧光素异硫氰酸酯或藻红蛋白偶联的单克隆抗体对以下表面抗原进行染色：CD34，E-选择素（CD62E），ICAM-1（CD54）和VCAM-1（CD106）。将细胞与浓度为100至800 mg / L（克拉霉素和阿奇霉素）和200至1,200 mg / L（红霉素）的抗生素一起孵育。这些浓度在治疗条件下在输注部位发生。将细胞温育2小时，并且在没有测试化合物的另外的22小时的培养时间之后进行分析。观察到所有四种抗原的表达均显着增强，最明显的是800 mg / L（红霉素），600 mg / L（阿奇霉素）和400 mg / L（克拉霉素）。浓度为800 mg / L的红霉素培养基引起CD34（+6％），E-选择素（+5％），ICAM-1（+ 14％）和VCAM-1（+ 5％）的表达增加。在较低浓度（600 mg / L）时，阿奇霉素可引起更强烈的促炎性抗原上调：CD34（+17％），E-选择素（+18％），ICAM-1（+ 27％）和VCAM-1（+17 ％）。在浓度为400 mg / L的情况下，克拉霉素诱导的效果与红霉素相似，是该浓度的两倍：CD34（+5％），E-选择素（+7％），ICAM-1（+ 23％）和VCAM-1（ + 4％）。 HUVEC的反应不如EA.hy 926细胞明显。参与内皮细胞和白细胞相互作用的细胞表面标志物被证明是研究三种大环内酯类药物促炎潜能差异的有用标志物。通过分析这些抗原在EA.hy 926细胞上的上调，对于其他药物也可以预测静脉炎的风险。

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