Integrins Specifically Associated with the Activated Phagocytic Receptor Complexes from Human U937 Macrophages and the Integrins of Human Blood

Receptor complexes are a key locus in the treatment of disease and pain but remain difficult to isolate. The Fc receptor binds to IgG presented on micro particles and triggers phagocytosis that is required for the engulfment of bacteria and non-self, pathogen molecules. Circulating LDL may be converted to oxLDL that is recognized by scavenger or other innate receptors that trigger the production of free radicals and phagocytic engulfment. The Fc receptor complex was captured using its cognate ligand IgG bound to 2 micron polystyrene beads from live cells by live cell affinity receptor chromatography (LARC). Similarly, oxLDL was used to coat micro beads to isolate the oxLDL-scavenger receptor complexes. The ligand coated beads were presented to live human macrophage U937 cells and sampled over the course of cell binding and engulfment in saline experimental medium. The receptor complexes were isolated by disruption with a French press and sucrose gradient centrifugation. The receptor complexes were separated using a salt and acetonitrile gradient prior to digestion with trypsin. The peptides were analyzed by LC-ESI MS/MS using a linear ion trap (Thermo) with the SEQUEST algorithm. In addition ligand affinity chromatography was performed using IgG, oxLDL or anti CD36 microbeads incubated with crude extracts. Controls included uncoated, or ligand coated, beads incubated in crude extract and/or used experimental medium. Certain integrins were shown to be specifically associated with IgG versus oxLDL and/or control treatments. The functional requirement for these integrins was tested using silencing RNA and quantitative assays of phagocytosis using laser scanning confocal microscopy. For the first time we show a small but statistically significant role for integrins in IgG-Fcγ receptor mediated phagocytosis.