Intraocular in vivo imaging of activated T-lymphocytes expressing green-fluorescent protein after stimulation with endotoxin.
BACKGROUND
Intravital microscopy allows imaging of specific cell populations in vivo. The value of this technique is well established, but would be enhanced if one could distinguish functional states of cells in vivo. Interleukin-2 (IL-2) is expressed upon stimulation of T-cells and is a commonly used marker for T-cell activation. This study tests the use of enhanced green fluorescent protein (GFP) as a reporter gene for interleukin-2 (IL-2) expression in vivo.


METHODS
Characterization of mice that have the GFP gene under the control of IL-2 regulatory sequences has previously been published. Uveitis was induced by injection of E. coli endotoxin into the vitreous of these IL-2/GFPki transgenic mice. Four hours later, 3 microg of recombinant 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 mm2 was evaluated.


RESULTS
Eyes with endotoxin-induced uveitis had cells that expressed GFP and were identifiable by intravital microscopy. The fluorescent 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 undetectable at baseline to 0.5 cells/mm2 at 6 h and 1.3 cells/mm2 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


CONCLUSIONS
GFP is commonly used as a reporter gene for in vitro expression assays. The results presented here document that transgenic mice with GFP under the control of IL-2 regulatory elements can be used with intravital microscopy for in vivo expression assays that allow detection of activated T-cells at multiple time points within the same animal. This provides a novel method for temporal and spatial studies on the state of cell activation in inflammatory responses.
Becker
M D
MD
Universitäts-Augenklinik. Heidelberg, Germany. matthias.becker@urz.uni-heidelberg.de
Crespo
S
S
Martin
T M
TM
Planck
S R
SR
Naramura
M
M
Rosenbaum
J T
JT
D000818:Animals; D000867:Anterior Chamber / Q000187:drug effects; D004731:Endotoxins; D004926:Escherichia coli; D005260:Female; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism*; D007376:Interleukin-2 / Q000008:administration & dosage / Q000378:metabolism; D007498:Iris / Q000276:immunology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008213:Lymphocyte Activation; D018655:Lymphocyte Count; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D014605:Uveitis / Q000139:chemically induced / Q000276:immunology
D004731:Endotoxins; D007202:Indicators and Reagents; D007376:Interleukin-2; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins