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*
Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, VA 22908;
Klinik und Poliklinik für Anästhesiologie und operative Intensivmedizin, Westf. Wilhelms-Universität Münster, Münster, Germany; and
Cardiovascular Research Center, University of Virginia Health Sciences Center, Charlottesville, VA 22908
Intravital microscopy allows detailed analysis of leukocyte
trafficking in vivo, but fails to identify the nature of leukocytes
investigated. Here, we describe the development of a CD2-enhanced green
fluorescence protein (EGFP)-transgenic mouse to characterize lymphocyte
trafficking during inflammation in vivo. A CD2-EGFP plasmid construct
including the CD2 promoter, the EGFP transgene, and the CD2 locus
control region was injected into B6CBA/F1 pronuclei.
EGFP+ offspring were backcrossed into C57BL/6 mice for six
generations. Flow cytometry demonstrated that all peripheral blood
EGFP+ cells were positive for CD2 and negative for the
granulocyte Ag Ly 6-G (GR-1). EGFPhigh cells stained
positive for CD2, CD3, CD8, TCR 
-chain, and NK1.1 but did not
express the B cell and monocyte markers CD45RA, CD19, and CD11b. In
vitro stimulation assays revealed no difference in lymphocyte
proliferation and IL-2 secretion between EGFP+ and
EGFP- mice. Intravital microscopy of untreated or
TNF-
-treated cremaster muscle venules showed EGFP+ cells
in vivo, but these cells did not roll or adhere to the vessel wall. In
cremaster muscle venules treated with both TNF- and IFN-
,
EGFPhigh cells rolled, adhered, and transmigrated at a
rolling velocity slightly higher (11 µm/s) than that of neutrophils
(10 µm/s). Blocking 4 integrin with a mAb increased
rolling velocity to 24 µm/s. These findings show that
CD8+ T cells roll in TNF-/IFN--pretreated vessels in
vivo via an 4 integrin-dependent
pathway.
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