Phagocytic receptor type affects phagosome maturation, gene expression and phagosome protein composition in macrophages. Mus musculus

The receptors engaged during phagocytic particle uptake determine the signalling events that occur during phagosome formation and maturation.However, pathogens generally have multiple ligands, making it difficult to dissect the roles of individual receptors in these processes. Here,we used latex beads coupled to single ligands, focusing primarily on IgG, mannan and avidin, and for some experiments on LPS, and monitored 1. phagocytic uptake rates, 2. fusion with lysosomal compartments, 3.macrophage gene expression 4. proteomic composition of isolated phagosomes and 5. the binding of phagosomes to actin filaments. Phagosomes formed in response to mannan fused with lysosomes earlier after uptake than the ones formed by the other ligands. The pattern of gene expression and the protein composition of isolated phagosomes showed that each bead ligand upregulated a distinct pattern of genes and led to a different composition of phagosomes. Furthermore, isolated phagosomes also exhibited differences in binding to F-actin in vitro.These data argue that activation of each receptor initiates a specific signature of signalling events that last many hours and influences several phagocytosis functions. Keywords: receptor-ligand interactions Overall design: J774.A1 cells (mouse macrophage-like cell line) were seeded onto 6-well plates one day before the experiment. Duplicates were incubated with serum-free DME medium containing 0.01 % latex beads of 1 µm diameter coupled to the following ligands: avidin (Av), Fc fragment of mouse IgG (Fc), lipopolysaccharides from Klebsiella pneumoniae (LPS) and mannan from Saccharomyces cerevisae (Man) for 30 and 60 minutes. After incubation samples as well as untreated controls in duplicates were washed twice in PBS and total RNA was extracted using RNAeasy kit (Qiagen) following manufacturer's instructions. Each sample was hybridized to CodeLink Mouse whole genome bioarray slides (Amersham). This study was intended to analyze the role of receptor-ligand interactions on phagosome maturation and gene expression after receptor-mediated phagocytosis in macrophages. Note that CodeLink EXP v4.0-processed data are represented in the Sample tables; GeneSpring-processed data are linked as a supplementary file at the foot of this record.