A mouse model of yellow fluorescent protein (YFP) expression in hematopoietic cells to assess leukocyte-endothelial interactions in the microcirculation

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 Ly6Chigh F4/80+ cells were YFPhigh while 55 ± 1% of Ly6Clow F4/80+ cells were YFPlow supporting the use of c-fmsYFP 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-fmsYFP+ mice, we evaluated leukocyte adhesion, rolling and local shear stress responses in the cremasteric endothelium 4 h following administration of TNFα. TNFα 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-fmsYFP+ 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 (r2 = 0.20, p < 0.05) was seen in the cremasteric microcirculation. Taken together, these data demonstrate the use of c-fmsYFP+ mice in identifying distinct monocyte subsets and highlight the potential of this model for real-time monocyte-endothelial interactions using intravital microscopy.