Degradation of phagosomal components in late endocytic organelles

Phagosomes are formed when phagocytic cells ingest particles such as bacteria, viruses or synthetic beads of different kinds. The environment within the phagosome gradually changes to generate degradative conditions. These changes require multiple interactions between the maturing phagosomes and the endocytic and the biosynthetic pathway. The phagosomes probably communicate with endocytic organelles by a transient fusion event, often referred to as the 'kiss-and-run' hypothesis. We have studied the role of endocytic organelles in the phagocytic pathway of J774 cells, a mouse macrophage cell line. We have used magnetic Dynabeads coated with 125ITC-IgG and 125ITC-OVA as phagocytic probes and were able to isolate the phagosomal fraction by means of a magnet. To separate lysosomes from other organelles in the endocytic pathway we allowed the cells to endocytose a pulse of colloidal gold particles complexed with ovalbumin. By combining this density shift technique with subcellular fractionation of a postnuclear supernatant in Percoll gradients we could isolate three endocytic fractions corresponding to early endosomes (the light Percoll fraction), late endosomes (the dense Percoll fraction) and lysosomes (the gold fraction). We observed that the proteins linked to the ingested beads are initially cleaved in the phagosomes. This cleavage is inhibited by leupeptin, a thiol-protease inhibitor, and requires an acidic environment. However, efficient communication between the phagosomes and the endocytic pathway leads to the transfer of dissociated phagocytosed peptides of different sizes to late endosomes and lysosomes for further processing. Consequently, the late endosomes and the lysosomes may be involved in the degradation of phagocytosed compounds.