4D intravital microscopy uncovers critical strain differences for the roles of PECAM and CD99 in leukocyte diapedesis

Abstract

Sullivan DP, Watson RL, Muller WA. 4D intravital microscopy uncovers critical strain differences for the roles of PECAM and CD99 in leukocyte diapedesis. Am J Physiol Heart Circ Physiol 311: H621-H632, 2016. First published July 15, 2016 doi:10.1152/ajpheart.00289.2016.-Leukocyte transendothelial migration (TEM) is an essential component of the inflammatory response. In vitro studies with human cells have demonstrated that platelet/endothelial cell adhesion molecule (PECAM) functions upstream of CD99 during TEM; however, results in vivo with mice have been apparently contradictory. In this study we use four-dimensional (4D) intravital microscopy to demonstrate that the site and order of function of PECAM and CD99 in vivo are dependent on the strain of mice. In FVB/n mice, PECAM functions upstream of CD99, as in human cells in vitro, and blocking antibodies against either molecule arrest neutrophils before they traverse the endothelium. However, in C57BL/6 mice, PECAM and CD99 appear to function at a different step, as the same antibodies arrest leukocyte migration through the endothelial basement membrane. These results are the first direct comparison of PECAM and CD99 function in different murine strains as well as the first demonstration of the sequential function of PECAM and CD99 in vivo.CD99; diapedesis; inflammation; intravital microscopy; platelet/endothelial cell adhesion molecule NEW & NOTEWORTHYThe data presented in this study help clarify seemingly discrepant findings in the field involving two of the common mouse strains used to study inflammation, particularly regarding the roles of PECAM and CD99 in leukocyte transendothelial migration.INFLAMMATION is part of the host's immune response to tissue damage. It involves the local delivery of leukocytes to sites of damage via a series of adhesive interactions with the endothelial cells (ECs) lining the postcapillary venules. The committed step of this process is transendothelial migration (TEM), or diapedesis, during which the leukocytes squeeze between adjacent ECs or traverse the body of an intact EC. Platelet/EC adhesion molecule (PECAM) and CD99 are two junctional adhesion molecules known to be critical for TEM (reviewed in Refs. 13 and 19). Human ECs in vitro have been used to show that PECAM functions upstream of CD99 to regulate leukocyte TEM; inhibiting PECAM function arrests leukocytes along the apical surface of the endothelium, whereas blocking CD99 arrests cells partway through EC junctions (16,23,34).In contrast to in vitro studies with human cells, in vivo studies of PECAM and CD99 in diapedesis have provided disparate results. PECAM blockade in vivo has been shown to inhibit leukocyte transmigration in all mouse strains tested except C57BL/6 mice and to arrest leukocytes along the endothelium in FVB/n mice, replicating findings in human cells in vitro. In contrast, in PECAM-deficient C57BL/6 mice, there is a transient defect in crossing the endothelial basement membrane (5, 9, 38) or no detectable effect on transmigration, depending on th...

Endothelial IQGAP1 regulates leukocyte transmigration by directing the LBRC to the site of diapedesis

“…For this assay, croton oil, a potent inflammatory stimulus, or carrier alone, was topically applied to both leaflets of one ear. After 5 h (to allow for sufficient recruitment of neutrophils), the animals were sacrificed and their ears harvested and processed for whole-mount confocal microscopy (Watson et al, 2015; Weber et al, 2015; Sullivan et al, 2016). Both chimeras recruited a similar number of neutrophils to the inflamed tissue.…”

“…All experiments in this study used mice between 8 and 12 wk old. The C57BL/6 LysM-eGFP mouse strain (obtained from Dr. Paul Kubes, University of Calgary, Calgary, Canada) was described previously (Faust et al, 2000; Sullivan et al, 2016). Briefly, the myelomonocytic cells of these mice were rendered fluorescent by the insertion of eGFP in the lysozyme M (LysM) locus, under control of the LysM promoter.…”

“…Croton oil–induced inflammation was performed essentially as described previously (Schenkel et al, 2004; Watson et al, 2015; Sullivan et al, 2016) using 20 µl of 0.9% croton oil in a 4:1 solution of acetone/olive oil (carrier) and allowing 5 h to induce inflammation. The contralateral ear was treated with carrier only.…”

“…Preparation of the cremaster muscle for visualization using fluorescence microscopy was performed essentially as described previously (Thompson et al, 2001; Sumagin and Sarelius, 2007; Woodfin et al, 2011; Sullivan et al, 2016). Briefly, male mice between 8 and 12 wk old were injected intrascrotally with 50 ng mouse recombinant IL-1β (R&D Systems) and 100 mg DyLight 550–conjugated nonblocking rat anti-mouse PECAM (clone 390) in a final volume of 100 µl 4 h before exteriorization and visualization of the cremaster muscle.…”

Endothelial cell-specific anticoagulation reduces inflammation in a mouse model of acute lung injury

“…In the current studies, the anticoagulants were administered intravenously, intratracheally or by nebulization, and no assessment of the mechanism of action was assessed. We have explored the impact of targeted anticoagulant therapy in a mouse model of ALI, using two strains of transgenic mice, CD31-TFPI-Tg and CD31-Hir-Tg, that expressed either a membrane-tethered hTFPI or a hirudin fusion protein, respectively, on all CD31-positive cells, including vascular ECs, monocytes, platelets, polymorphonuclear leukocytes (PMNs) and T cells [13][14][15][16] .…”