TY - JOUR
T1 - A CD2-green fluorescence protein-transgenic mouse reveals very late antigen-4-dependent CD8+ lymphocyte rolling in inflamed venules
AU - Singbartl, K.
AU - Thatte, J.
AU - Smith, M. L.
AU - Wethmar, K.
AU - Day, K.
AU - Ley, K.
PY - 2001/6/15
Y1 - 2001/6/15
N2 - 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 β-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-α-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-α and IFN-γ, EGFPhigh cells rolled, adhered, and transmigrated at a rolling velocity slightly higher (11 μm/s) than that of neutrophils (10 μm/s). Blocking α4 integrin with amAb increased rolling velocity to 24 μm/s. These findings show that CD8+ T cells roll in TNF-αIFN-γ-pretreated vessels in vivo via an α4 integrin-dependent pathway.
AB - 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 β-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-α-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-α and IFN-γ, EGFPhigh cells rolled, adhered, and transmigrated at a rolling velocity slightly higher (11 μm/s) than that of neutrophils (10 μm/s). Blocking α4 integrin with amAb increased rolling velocity to 24 μm/s. These findings show that CD8+ T cells roll in TNF-αIFN-γ-pretreated vessels in vivo via an α4 integrin-dependent pathway.
UR - http://www.scopus.com/inward/record.url?scp=0035876932&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0035876932&partnerID=8YFLogxK
U2 - 10.4049/jimmunol.166.12.7520
DO - 10.4049/jimmunol.166.12.7520
M3 - Article
C2 - 11390506
AN - SCOPUS:0035876932
SN - 0022-1767
VL - 166
SP - 7520
EP - 7526
JO - Journal of Immunology
JF - Journal of Immunology
IS - 12
ER -