Real-Time Identification of Leukocyte Subsets and Cell Surface Receptor Microdomains in the Microvasculature of Wild-Type and Sickle Cell Mice In Vivo.
Authors: Chiang Elaine Y. , Hidalgo Andres , Chang Jungshan , Frenette Paul S. 
Published: April 2020

Abstract
Intravital microscopy has contributed enormously to the recent mechanistic advances in leukocyte (WBC) trafficking. However, current methods have provided little knowledge about differential WBC subset behavior or the spatial redistribution of surface adhesion molecules during recruitment and activation. Here, we use digital high-speed multichannel fluorescence intravital videomicroscopy to identify WBCs and localize cell surface molecules. Adherent WBCs were ascertained using a series of near-simultaneous 4-channel images in randomly selected venular segments following the injection of low-dose (0.02–0.12 mg/kg) fluorescently labeled Mab against CD45, Gr-1, and F4/80. Preliminary experiments on FAC-sorted WBCs revealed that Gr–1+F4/80- cells were highly enriched in neutrophils (PMNs, ~99%), whereas the F4/80+ fraction was mostly monocytes (~70%) and the fraction negative for both markers was largely lymphocytes (~88%). In the cremaster muscle of C57BL/6 mice (n=6), 39 ± 4% of adherent CD45+ cells recruited in venules ~90 min following surgery were Gr–1+F4/80- (PMNs), and 45 ± 7% were F4/80+ (monocytes), while 18 ± 3% were Gr–1-F4/80- (lymphocytes). In contrast, the proportion of PMNs was significantly increased in cremasteric venules of sickle cell (SS) mice (Berkeley) (79 ± 7%, n=5; p&lt;0.001 compared to C57BL/6). Our previous studies have revealed that the interactions between erythrocytes (RBCs) and adherent WBCs correlate with vascular occlusion and mortality in SS mice (Turhan et al, PNAS, 2002). To determine which WBC subset can capture RBCs in vivo, we monitored RBC-WBC interactions in high-resolution brightfield movies acquired immediately after the capture of fluorescence images. We found that the vast majority of interactions (~93%) occurred with CD45+Gr–1+F4/80- PMNs. The high proportion of lymphocytes in systemic venules was unexpected. We thus analyzed directly lymphocyte subclasses with antibodies specific for B and T cell markers (B220 and CD4/CD8). The proportion of B and T cells was consistent with the above results (total ~20%), similar between C57BL/6 and sickle cell mice, with B cells predominating in both groups (70–76% of all lymphocytes). Since the bone marrow (BM) is a target of SS disease, we examined differential WBC recruitment in this vascular bed. In contrast to the cremasteric microcirculation, CD45+Gr–1-F4/80- lymphocytes represented the major WBC subset in both C57BL/6 and SS venules (49 ± 2% and 63 ± 5%, respectively.) Adherent CD45+B220+ B lymphocytes were significantly more numerous than CD45+CD4/CD8+ T lymphocytes in C57BL/6 (6-fold ↑, p&lt;0.001) and SS (9-fold ↑, p&lt;0.005) mice. We next evaluated the spatial distribution of LFA-1 and PSGL–1 on Gr–1+ WBCs recruited in TNF-α-stimulated cremasteric venules. We observed that PSGL–1 distribution was polarized in the great majority of adherent Gr–1+ cells (93 – 97%), whereas neither LFA–1 nor Gr–1 was polarized. The majority of PSGL–1 clusters was luminal rather than adjacent to the endothelium in both stationary and slowly migrating cells. In stationary cells, PSGL–1 appeared random in relation to the direction of blood flow. By contrast, in migrating cells, PSGL–1 redistribution was strongly associated with the direction of cell migration, with PSGL–1 clusters being almost exclusively located at the trailing edge. In summary, our results reveal a profound difference in the WBC subsets recruited in the cremastric and BM venules, and a powerful method to analyze the formation of discrete microdomains in vivo during cell activation.