Azithromycin, a pharmacological agent which selectively impairs some pathways of endocytosis : characterization, interests and mechanism of action

Endocytosis is required for a vast number of functions that are essential for the well being of the cell. Before the advent of electron microscopy and molecular biology, endocytosis were adopted to describe the uptake of large particles and extracellular medium respectively. However, the situation was more complex and three modes of endocytosis were described : (i) fluid-phase endocytosis, (ii) non-specific adsorptive endocytosis, and (iii) receptor-mediated endocytosis. In addition, multiple internalization mechanisms are clearly established, and among these are : (i) clathrin-dependent endocytosis, i. e. endocytosis mediated by small vesicles that have a morphologically characteristic coat made of complex proteins that include clathrin ; (ii) clathrin-independent endocytosis ; and (iii) phagocytosis, i. e. the uptake of large particular ligands including bacteria and apoptotic cells. A number of internalization mechanisms is described and several functions are ascribed to each. However, there is still a multitude of questions. In this context, pharmacological agents could be usefull to dissect endocytic and phagocytic pathways. Agents perturbing endocytosis are numerous. However, a few of these agents is really specific of one pathway and/or one step of endocytosis. In view of the lack of specific inhibitors, in particular of clathrin-independent endocytosis, we were interested by the macrolide antibiotic azithromycin (AZ), based on the facts that this drug : (i) has a marqued amphiphilic character and extensively accumulates in lysosomes of cultured fibroblasts ; (ii) induces a lysosomal phospholipidosis ; (iii) is able to bind to negatively charged bilayers at acidic pH ; and (iv) is able to perturb the fusion of phospholipid-overloaded lysosomes with horseradish peroxidase (HRP ; a tracer of fluid-phase endocytosis) containing endosomes (preliminary observation in electron microscopy). The first part of this study was performed to characterize the effect of AZ on fluid-phase endocytosis in rat foetal fibroblasts. AZ inhibited the uptake of HRP by fluid-phase endocytosis in fibroblasts in a time- and concentration-dependent fashion without affecting its decay (regurgitation and/or degradation). The AZ effect was additive to that of nocodazole, known to impair transport of solutes along the endocytic pathway. Cytochemistry showed a major reduction by AZ in the number of labeled-HRP endocytic vesicles at 5 min (endosomes) and 2 h (lysosomes). Within 3 h of exposure, AZ also caused the appearance of large and lightlucent electron-lucent vacuoles. Three days of treatment with AZ resulted into the accumulation of very large vesicles filled with pleiomorphic content, consistent with phospholipidosis. These vsicles were accessible to fluorescein-labeled bovine serum albumin (FITC-BSA) and intensively stained with filipin, indicating a mixed storage with cholesterol. The impairment of HRP endocytosis directly correlated with the amount of AZ accumulated by the cells, but not with the phospholipidosis induced by the drug. The proton ionophore monensin, which completely suppresses AZ accumulation, also prevented inhibition of HRP uptake. The aim of the second part was to evaluate if AZ was a specific inhibitor for fluid-phase endocytosis or not and was performed on J774 mouse macrophages. Pretreatment of macrophages by AZ, selectively inhibited fluid-phase endocytosis of HRP and lucifer yellow, but not phagocytosis of latex beads. AZ delayed sequestration of receptor-bound transferrin and peroxidase-anti-peroxidase immune complexe into cell-surface endocytic pits and vesicles, but did not slow down the subsequent rate of receptor-mediated endocytosis. AZ down-regulated cell surface transferrin-receptors, but not Fcγ-receptors, by causing a major delay in the accessibility of internalized transferrin-receptors to the recycling route, without slowing down sugsequent efflux, and resulting in redistribution of the surface pool to an intracellular pool. Acidotropic accumulation of AZ was associated with an extensive vacuolation of late endosomes/lysosomes, and these compartments became unaccessible to HRP and immune complexes, but not to latex beads. The inhibitory profile of AZ cannot be solely accounted for by vacuolation and interference with acidification, and the effect of AZ could occur by perturbing lateral mobility of receptors and/or budding of endocytic pits into endocytic vesicles. The third part of this study was thus performed to analyse interactions of AZ with artificial models and J774 macrophages and to evaluate the consequences of these interactions on both membrane properties involved in endocytosis and membrane endocytosis in itself. AZ directly interacted with membranes and reduced the mobility of phosphate heads but had no effect deeper within the bilayer. AZ did not induce membrane fusion, but perturbed the organisation of model membranes in domains, another membrane property involved in endocytosis. The consequece of AZ-membrane interaction on membrane endocytosis in itself was evaluated by using three membrane tracers which differ by their localisation inside the membrane and their intracellular trafficking : 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosyl phosphocholine (C6-NBD-SM), trimethylammonium-diphenyl-hexatriene (TMA-DPH) and N-(lissamine rhodamine B sulfonyl) diacyl phosphatidyl ethanolamine (N-Rh-PE). AZ decreased J774 surface-incorporation of these membrane tracers, and slowed down membrane accumulation destined to lysosomes, evaluated by using N-Rh-PE, but had no effect on C₆-NBD-SM and TMA-DPH internalization. AZ decreased the J774 plasmamembrane fluidity, measured by fluorescence polarisation of TMA-DPH and confirmed by the absence of patches of fluorescent concanavalin A. We suggested that AZ was able to interact with membranes and to selectively perturb the organisation, fluidity and endocytic capacity of specific domains of the plasma membrane.Thèse de doctorat en sciences pharmaceutiques (FARM 3) -- UCL, 200