Leukocyte-endothelial interactions are dependent on molecular signals displayed on the EC surfaces. As the immune response is a dynamic process, the molecular signals are expressed in a spatially as well as temporally specific manner [37
]. Traditional methods such as immunohistochemistry require tissue extraction/processing and provide only snapshots of endothelial expression at single time points. In addition, immunohistochemical methods yield little three-dimensional information because tissue is cut into thin sections. Intravital microscopy has proved to be a powerful method for visualizing leukocyte trafficking in vivo and has led to a wealth of new understanding on how circulating cells home to different tissue sites under normal and diseased conditions. However, endothelial molecular expression is usually not directly visualized in these studies. Instead, the contributions of specific cell adhesion molecules to the leukocyte-endothelial interaction are determined indirectly by comparing the leukocyte trafficking patterns before and after treatment with an interfering antibody or peptide directed against the adhesion molecules [18
], or by comparing wild-type and knockout animals [38
]. Although these functional studies are essential, they do not reveal the detailed spatial distribution of the molecules under investigation. Newer methods image molecular expression in vivo by labeling endothelial cell markers with immunoconjugates of magnetic resonance [39
], ultrasound [41
], or radionuclide contrast agents [33
], but these methods lack the spatial resolution to visualize single cells and small blood vessels.
Here we demonstrate that the temporal and spatial relationships of vascular endothelium molecular expression can be visualized directly using the method of in vivo immunofluorescence microscopy. Through optical sectioning, this method provides three-dimensional information on the architecture of the vasculature, and because it is noninvasive in nature, the same animals can be imaged repeatedly to yield temporal data. Endothelial cell surface molecules are labeled with fluorescent antibodies injected into the circulation, and the vasculature of the skin or the eye can be imaged noninvasively in live animals over time. Other vascular beds, as in bone marrow [36
], can be imaged with minimum tissue manipulation. We have used primary antibody concentrations of 0.5–1.0 mg/kg, which does not appear to be saturating because the fluorescence signal increases with increasing antibody dosage (data not shown). In addition, we have shown in another work [36
] that leukemic and progenitor/stem cell homing and engraftment to bone marrow vasculature are not affected by the presence of antibodies against E-selectin and SDF-1 at similar concentrations, indicating minimal perturbation to cell function. Falati et al. [26
] demonstrated the formation of thrombii in the presence of primary antibody concentrations nearly 10 times higher than used in the current work.
The use of optical sectioning techniques such as confocal or multiphoton microscopy greatly enhances the capacity to detect the weak immunofluorescence signal amid the stronger tissue autofluorescence background. Imaging depth is limited by tissue scattering to about 150 μm in the skin, but this depth is sufficient for visualizing most of the vascular structures in the mouse skin as well as the marrow of the flat bone of the skull. In addition, the fast image-acquisition speed (up to video rate at 30 frames per second) allows us to image moving targets such as rolling leukocytes as in standard intravital microscopy [43
]. For stationary targets, such as endothelial cells, the imaging speed makes it possible to survey relatively large tissue volumes (e.g., most of the mouse ear).
Using this method, we are able to detect (1) PECAM-1 expression in the vasculature of skin, retina, and choroid; (2) E-selectin constitutive expression in the small postcapillary venules with diameters less than 50 μm in normal mouse skin and upregulation in large venules (>50 μm) in skin after LPS treatment; (3) upregulation of E-selectin in conjunctiva after LPS induction; and (4) P-selectin constitutive expression in normal mouse skin both in small (<50 μm) and larger (>50 μm) venules.
Our results agree with the finding that E-selectin is expressed in the skin of the mouse under normal, non-inflammatory conditions. In addition, in vivo immunufluorescence confocal microscopy revealed that E-selectin in skin is expressed in venules of varying sizes in the quiescent and induced states, with constitutively expressed E-selectin restricted to small postcapillary venules. The data presented here are consistent with the argument that E-selectin expression in the skin may be specifically designed to enable memory T cells expressing cutaneous lymphocyte antigen (CLA) to roll slowly and perform immune surveillance [44
]. P-selectin expression in normal skin is also restricted to the venules, but in contrast to E-selectin is found in both small and large venules. The pattern of EC molecular expression controls the nature of an immune response at a given site [45
]. Antibody blocking studies using in vivo fluorescence microscopy may be used to dissect these relationships.
In summary, this article demonstrates the use of in vivo immunofluorescence microscopy to directly characterize EC surface marker expression in mouse skin and eye under normal and inflamed conditions. The major advantage of the live imaging approach is that (1) spatial and temporal relationships in the expression of individual cell markers can be determined using multichannel imaging; (2) as a result, the three dimensional architecture of the vasculature can be discerned, and specific cell populations can be dynamically followed; and (3) individual experimental animals can be imaged repetitively over several days or weeks to follow the time course of disease progression or response to therapy. This method will be particularly useful for studying processes such as inflammation, angiogenesis, atherogenesis, and diabetic retinopathy, which are closely associated with EC activation, proliferation, or dysfunction.