Abstract
In this study, we describe the use of intravital microscopy in a transgenic mouse model expressing yellow fluorescent protein (YFP) under the control of a monocyte specific promoter c-fms (CD115) to track and quantify specific leukocyte subsets. Flow cytometry on peripheral and bone marrow leukocytes revealed that YFP was predominantly expressed by CD11a(+), CD11b(+), and CD14(+) monocytes. In the bone marrow, 67+/-4% of Ly6C(high) F4/80(+) cells were YFP(high) while 55+/-1% of Ly6C(low) F4/80(+) cells were YFP(low) supporting the use of c-fms(YFP) expression as a marker of monocyte lineage. 70+/-7% of CD11b(+) F4/80(+) Ly6C(+) ("triple positive") cells expressed YFP. To assess leukocyte-endothelial interactions in YFP(+) cells in c-fms(YFP+) mice, we evaluated leukocyte adhesion, rolling and local shear stress responses in the cremasteric endothelium 4 h following administration of TNFalpha. TNFalpha resulted in a five-fold increase in adhesion of YFP(+) cells to the endothelium and provided superior discriminative ability in assessing rolling and adhesion events when compared with bright field microscopy. Additionally, when compared with Rhodamine-6G labeled leukocytes or GFP(+) cells in mice transplanted with green fluorescent protein (GFP) positive bone marrow, the level of detail observed in the c-fms(YFP+) was greater, with both GFP(+) and YFP(+) cells demonstrating superior signal to noise compared to bright field microscopy. A weak positive linear correlation between wall shear stress and YFP(+) cell adhesion (r(2)=0.20, p<0.05) was seen in the cremasteric microcirculation. Taken together, these data demonstrate the use of c-fms(YFP+) mice in identifying distinct monocyte subsets and highlight the potential of this model for real-time monocyte-endothelial interactions using intravital microscopy.