A CD2-green fluorescence protein-transgenic mouse reveals very late antigen-4-dependent CD8+ lymphocyte rolling in inflamed venules.
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). EGFP(high) cells stained positive for CD2, CD3, CD8, TCR beta-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-alpha-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-alpha and IFN-gamma, EGFP(high) cells rolled, adhered, and transmigrated at a rolling velocity slightly higher (11 microm/s) than that of neutrophils (10 microm/s). Blocking alpha4 integrin with a mAb increased rolling velocity to 24 microm/s. These findings show that CD8+ T cells roll in TNF-alpha/IFN-gamma-pretreated vessels in vivo via an alpha4 integrin-dependent pathway.
Singbartl
K
K
Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.
Thatte
J
J
Smith
M L
ML
Wethmar
K
K
Day
K
K
Ley
K
K
D000818:Animals; D015703:Antigens, CD / Q000502:physiology; D000954:Antigens, Surface / Q000096:biosynthesis; D018801:CD2 Antigens / Q000096:biosynthesis / Q000235:genetics*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000473:pathology*; D002465:Cell Movement / Q000235:genetics / Q000276:immunology*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007249:Inflammation / Q000235:genetics / Q000276:immunology; D039441:Integrin alpha4; D039041:Integrin alpha4beta1; D016023:Integrins / Q000502:physiology*; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D008213:Lymphocyte Activation / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018715:Microscopy, Video; D018482:Muscle, Skeletal / Q000098:blood supply; D016201:Receptors, Lymphocyte Homing / Q000502:physiology*; D014699:Venules / Q000276:immunology* / Q000473:pathology*
D015703:Antigens, CD; D000954:Antigens, Surface; D018801:CD2 Antigens; D039041:Integrin alpha4beta1; D016023:Integrins; D008164:Luminescent Proteins; D016201:Receptors, Lymphocyte Homing; D039441:Integrin alpha4; D049452:Green Fluorescent Proteins