Abstract
Purpose:
Prior studies have shown αvβ5 regulates phagocytosis in TM cells (Gagen et al. 2013. IOVS. 54:5000-11) and that integrin crosstalk between αvβ3 and αvβ5 integrins inhibited phagocytosis. The goal of this study was to determine if αvβ5 integrin-mediated phagocytosis in TM cells involves Rac1.
Methods:
To determine if phagocytosis in TM cells was a Rac1-mediated process, immortalized TM-1 cells were pretreated for 30 minutes with the Rac1 inhibitors 50 µM EHop-016, 60 µM NSC23776, and 10µM EHT 1864. The Rac1 inhibitor EHop-016 prevents the GEFs Vav1 and Vav2 from activating Rac1 while NSC23776 and EHT 1864 prevent binding of the GEFs Trio and Tiam1 to Rac1. The phagocytic activity of control and Rac1 inhibitor-treated TM-1 cells was then determined by challenging TM-1 cells with pHrodo-labeled S. aureus bioparticles for 4 hrs. FACs and fluorescence microscopy were used to evaluate the level of phagocytic activity. Phase microscopy was used to evaluate cell morphology. RT-qPCR was done to determine the level of Vav, Tiam1 and Trio expression in TM cells.
Results:
By phase microscopy, none of the Rac1 inhibitors affected cell morphology. Both the Rac1-treated and control cells appeared well spread and did not show any signs of cell rounding or contraction. Fluorescence microscopy showed that cells pretreated with 50 µM Ehop-016 showed a significant reduction in the uptake of pHrodo-labeled bioparticles compared to control cells and cells treated with the other Rac1 inhibitors. FACs analysis supported the fluorescence microscopy studies and showed that phagocytosis in TM-1 cells pretreated with 50 µM EHop-016 was reduced by 75% compared to control cells. Phagocytic activity in cells pretreated with 60 µM NSC23776 or 10µM EHT 1864 was not different from that of control cells. RT-qPCR showed that TM-1 cells expressed Vav2 and Trio. Low levels of Vav3 were also detected. Tiam1 was not detected.
Conclusions:
The data suggests that phagocytosis in TM-1 cells involves an αvβ5 integrin/Vav2/Rac1 signaling pathway.
Keywords: 735 trabecular meshwork •
645 phagocytosis and killing