Intraocular in vivo imaging of activated T-lymphocytes expressing green-fluorescent protein after stimulation with endotoxin

Background: Intravital microscopy allows imaging of specific cell populatio ns in vivo. The value of this technique is well established, but would be e nhanced if one could distinguish functional states of cells in vivo. Interl eukin-2 (IL-2) is expressed upon stimulation of T-cells and is a commonly u sed marker for T-cell activation. This study tests the use of enhanced gree n fluorescent protein (GFP) as a reporter gene for interleukin-2 (IL-2) exp ression in vivo. Methods: Characterization of mice that have the GFP gene u nder the control of IL-2 regulatory sequences has previously been published . Uveitis was induced by injection of E. coli endotoxin into the vitreous o f these IL-2/GFP(ki) transgenic mice. Four hours later, 3 mug of recombinan t mouse IL-2 was injected into the anterior chambers of one group of mice. In vivo imaging of infiltrating cells in the iris stroma was performed with fluorescence microscopy at 6, 24, 48, and 72 h after endotoxin injection. The absolute number of fluorescent cells per mm(2) was evaluated. Results: Eyes with endotoxin-induced uveitis had cells that expressed GFP and were i dentifiable by intravital microscopy. The fluorescent cells were exclusivel y seen in the subset of cells that had infiltrated the iris stroma or arres ted along the vascular endothelium. The number of GFP-positive infiltrating cells in the iris increased from undetectable at baseline to 0.5 cells/mm( 2) at 6 h and 1.3 cells/mm(2) at 72 h. The animals that received endotoxin as well as IL-2 tended to have more GFP-positive cells at the 48-h and 72-h time points, but these differences were not statistically significant Conc lusions: GFP is commonly used as a reporter gene for in vitro expression as says. The results presented here document that transgenic mice with GFP und er the control of IL-2 regulatory elements can be used with intravital micr oscopy for in vivo expression assays that allow detection of activated T-ce lls at multiple time points within the same animal. This provides a novel m ethod for temporal and spatial studies on the state of cell activation in i nflammatory responses.