Maye I, de Fraissinette A, Cruz-Orive L M, Vonderscher J, Richter F, Gehr P
Institute of Anatomy, University of Bern, Switzerland.
Cell Mol Life Sci. 1997 Aug;53(8):689-96. doi: 10.1007/s000180050089.
The aim of this study was to develop an in vitro model to estimate the clearance of pulmonary administered cyclosporine A (CsA). To do this we estimated the volume of CsA particles phagocytosed by alveolar macrophages (AM) lavaged from hamsters. AM were cultured with CsA particles at two doses of particles (0.1 mg or 0.5 mg) and at three incubation times (1 h, 6 h or 24 h). The AM were also incubated with or without latex particles. After incubation, AM were processed for light and electron microscopy and the mean volume of phagocytosed particles was estimated stereologically from micrographs of the cells. Here, however, the CsA particles were dissolved during the embedding process and only their negative images (vacuoles) could be detected. An indirect method was therefore developed. The volume of cytoplasmic vacuoles (called 'background' vacuoles) was estimated in control macrophages (without particles or with latex particles and subtracted from the total volume of vacuoles in macrophages incubated with CsA, which gave the volume of phagocytosed CsA. The volume of the 'background' vacuoles remained constant in all study conditions. At a dose of 0.1 mg CsA the volume phagocytosed per macrophage was 13.83 microns3 at 1 h, 8.43 microns3 at 6 h and 4.50 microns3 at 24 h. At a dose of 0.5 mg CsA, the volume phagocytosed varied from 26.59 microns3 at 1 h, to 4.13 microns3 at 6 h and 49.10 microns3 at 24 h. These results show no statistically significant dependence on time for either dose, and a statistically significant dose effect only at 24 h. With latex particles, the phagocytosed volume increased significantly with time and dose and was significantly higher than for CsA particles. This study showed that CsA particles are phagocytosed by AM from hamsters but to a lesser extent than latex particles. This difference could be correlated with physical properties, i.e. a difference between particle size and shape and/or chemical properties, latex particles being inert and CsA particles being peptidic. Moreover, different surface receptors on AM could be involved in the process of phagocytosis of CsA and latex particles.
本研究的目的是建立一种体外模型，以估计经肺部给药的环孢素A（CsA）的清除率。为此，我们估计了从仓鼠肺泡灌洗获得的肺泡巨噬细胞（AM）吞噬的CsA颗粒的体积。将AM与两种剂量的CsA颗粒（0.1 mg或0.5 mg）以及三个孵育时间（1小时、6小时或24小时）一起培养。AM还分别在有或没有乳胶颗粒的情况下进行孵育。孵育后，对AM进行光镜和电镜处理，并从细胞显微照片中立体地估计吞噬颗粒的平均体积。然而，在此过程中CsA颗粒在包埋过程中溶解，只能检测到它们的负像（空泡）。因此开发了一种间接方法。在对照巨噬细胞（无颗粒或有乳胶颗粒）中估计细胞质空泡（称为“背景”空泡）的体积，并从与CsA一起孵育的巨噬细胞的空泡总体积中减去该体积，从而得出吞噬的CsA的体积。在所有研究条件下，“背景”空泡的体积保持恒定。在0.1 mg CsA剂量下，每个巨噬细胞吞噬的体积在1小时时为13.83立方微米，6小时时为8.43立方微米，24小时时为4.50立方微米。在0.5 mg CsA剂量下，吞噬的体积从1小时时的26.59立方微米变化到6小时时的4.13立方微米，24小时时为49.10立方微米。这些结果表明，两种剂量下吞噬体积在统计学上均与时间无显著相关性，仅在24小时时存在统计学上显著的剂量效应。对于乳胶颗粒，吞噬体积随时间和剂量显著增加，且显著高于CsA颗粒。本研究表明，CsA颗粒可被仓鼠的AM吞噬，但程度低于乳胶颗粒。这种差异可能与物理性质相关，即颗粒大小和形状的差异和/或化学性质，乳胶颗粒是惰性的，而CsA颗粒是肽类的。此外，AM上不同的表面受体可能参与了CsA和乳胶颗粒的吞噬过程。
