Visualization of monocyte recruitment into atherosclerotic arteries using fluorescent labelling

It has been shown that monocytes are present in early atherosclerotic lesions and in mechanically-injured arterial intima, but direct morphological tracing of specific leukocyte populations into such areas has been lacking. A method for FITC-labelling of leukocytes was therefore evaluated for monocyte studies. Monocyte (95% pure) populations were isolated from blood by counterflow centrifugation and labelled by incubation with free fluorescein isothiocyanate 1-hydrochloride (FITC) in Hank's balanced salt solution. FITC-labelled monocytes showed glass adherence, spreading and migration, as well as acid phosphatase positivity and phagocytosis for up to 20 days in tissue culture. For in vivo experiments, hypercholesterolemic (H) and normal (N) swine were bled repeatedly, and monocyte populations were isolated, labelled and reinjected. Labelled cells were found in blood samples. Animals were killed after 9 days, and formaldehyde-fixed and frozen samples of aortae were studied en face and/or sectioned and examined microscopically under fluorescence. FITC-labelled leukocytes could be found adherent to sites of thickened intima but not to normal areas. Labelled cells were also detected within atherosclerotic lesions. These results show the feasibility of the labelling technique and provide direct visualization of monocyte recruitment from the blood into atherosclerotic and lesion-prone areas.