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 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.
Kampfrath
Thomas
T
Davis Heart and Lung Research Institute, The Ohio State University College of Medicine, 473 W. 12th Avenue, Room 110, Columbus, OH 43210, USA.
Deiuliis
Jeffrey A
JA
Moffatt-Bruce
Susan D
SD
Anderson
Jeffrey
J
Sun
Qinghua
Q
Wood
Karen
K
Ostrowski
Michael C
MC
Rajagopalan
Sanjay
S
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism; D016026:Bone Marrow Transplantation; D002448:Cell Adhesion / Q000502:physiology; D002450:Cell Communication; D019070:Cell Lineage; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000502:physiology; D005434:Flow Cytometry; D006410:Hematopoiesis / Q000502:physiology; D036904:Leukocyte Rolling / Q000502:physiology; D008164:Luminescent Proteins / Q000378:metabolism*; D016131:Lymphocyte Subsets / Q000166:cytology* / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008833:Microcirculation / Q000502:physiology; D018715:Microscopy, Video; D023421:Models, Animal; D018482:Muscle, Skeletal / Q000098:blood supply
D015415:Biomarkers; D008164:Luminescent Proteins
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