VV 406
Intraocular in vivo imaging of activated T lymphocytes expressing green fluorescent protein (GFP)
M. D. Becker, S. Crespo, S. R. Planck, M. Naramura*, J. T. Rosenbaum
Purpose: Intravital microscopy allows imaging of specific cell populations in vivo. This technique has proven to be valuable, but would benefit from the ability to distinguish functional states of cells in vivo. By inserting the cDNA for green fluorescent protein (GFP) after the promotor region of the interleukin-2 (IL-2) gene [Immunity (1998) 9: 209] a specific marker is created for T-cells that have been activated.
Method: Uveitis was induced by injection of E. coli endotoxin into the vitreous of IL-2/GFP transgenic mice. 4h later 3 µg of recombinant mouse IL-2 was injected into the anterior chamber. In vivo imaging of infiltrating cells in the iris stroma was performed with fluorescence microscopy at 6, 24, 48, and 72 hours after endotoxin injection. The absolute number of fluorescent cells per mm2 was evaluated.
Result: Activated T-cells expressing GFP were detected and visualized by intravital microscopy. Activated cells were exclusively seen in the subset of cells that had infiltrated the iris stroma or arrested along the vascular endothelium. The number of GFP-positive infiltrating cells in the iris increased from 1.5 cells/mm2 at 6 hours to 5.7 cells/mm2 at 72 hours.
Conclusion: GFP expression in these genetically altered mice allows detection of activated T-cells in vivo at multiple time points within the same animal. This study describes a novel method for determining the state of cell activation in inflammatory responses.
Casey Eye Institute, Oregon Health Sciences University, Portland, OR
*National Institutes of Health, Bethesda, MD