Abstract
Purpose: :
To investigate the effect of chronic phagocytic stress on lysosomal function in cultured porcine trabecular meshwork (PTM) cells.
Methods: :
Three independent cultures of PTM cells were challenged with 1 x 107 particles of different phagocytic ligands (FITC-labeled E.coli and fluorescent latex beads) for 2, 5, and 10 days. Phagocytic capacity was evaluated by fluorescence microscopy. Lysosomal content was quantified by flow cytometry using lysotracker red. Co-localization of phagocytic ligands within the lysosomal compartment was monitored by confocal microscopy. Ultrastructural analysis was performed by transmission electron microscopy. Kinetics of phagosome maturation as a function of phagosome/lysosome fusion was monitored using E.coli labeled with the pH sensitive dye pHrodo. Cathepsin levels were quantified by qPCR and WB analysis. Cathepsin activities were evaluated using fluorogenic substrates (z-FR-AMC, z-RR-AMC, z-GPR-AMC, z-VVR-AMC, CTSD/E substrate).
Results: :
PTM cells showed an innate capacity to ingest large amounts of inert and biologically active particles, without significantly affecting cell viability; only slight cytotoxicity was observed with E. coli at Day 10. Electron micrographs revealed the presence of multivesicular bodies, phagolysosomes, as well as intracellular and extracellular membranous figures following phagocytic challenge. Co-localization of ingested particles within lysosomes and increased lysosomal content (40.17% ± 5.67, p<0.05, n=3) were observed with E. coli, but not with beads. Kinetics analysis showed that E. coli reached the lysosomal compartment within the first 2 hours. Phagocytosis of E. coli was significantly reduced in the absence of serum, suggesting Fc receptor-mediated phagocytosis. The mRNA and activity levels of several cathepsins (K, L, D, and B) and genes related with the autophagy/lysosomal pathway (ATG6, LAMP-2) were upregulated with phagocytic stress. The most striking changes were the sustained dramatic increase of cathepsin B expression (mRNA and protein levels; 2.64±0.5 fold, 5.03±1.46 fold, and 8.76±2.55 fold at day 2, 5, and 10, respectively; p<0.05, n=3) and cathepsin B activity (77.2±12.1%, 82.4± 7.6%, and 94.1±11.3% at day 2, 5, and 10, respectively; p<0.05, n=3) in E. coli challenged-cultures.
Conclusions: :
Our results indicate that although PTM cells are able to efficiently phagocytose different materials, the fate and elicited responses vary among the nature of the ingested particles. A better understanding of the phagocytic/lysosomal functions in TM cells is particularly important given the potentially critical role of ingestion/degradation of extracellular material in outflow pathway function.
Keywords: trabecular meshwork • phagocytosis and killing • anterior segment