Quantitation of L-selectin distribution on human leukocyte microvilli by immunogold labeling and electron microscopy.

Abstract

L-Selectin is a leukocyte cell adhesion receptor that contributes to neutrophil (PMN) rolling on activated endothelium at sites of idammation and mediates lymphocyte attachment to high endothelial venules in peripheral lymph nodes. Localization of this receptor to the tips of PMN and lymphocyte microvilli has been demonstrated. However, its distribution on these cells has not been quantified, and its localization on other leukocytes and the morphometry of microvilli on different leukocyte subpopulations have not been previously examined. In this study, PMN and mononuclear leukocytes were isolated from anticoagulated blood by dextran sedimentation and density centrifugation, fixed in 2% paraformaldehyde and 0.05% glutaraldehyde, immunogold-labeled for L-selectin, and embedded in Epon resin. The distribution of L-selectin was determined by counting gold particles on the plasma membrane of sectioned cells, and the surface microstructure of these cells was surveyed on two-dimensional transmission electron micrographs. On average, 78% of PMN, 72% of monocyte, and 71% of lymphocyte L-selectin was observed on the microvilli, with more Variance on lymphocytes than the other cell types. Typical PMN and monocyte sections had 26 microvilli, whereas typical lymphocyte sections had 23. Quantitation of the distribution of L-selectin and leukocyte surface topology offers a foundation from which to study the requirement of microvilli or microvillus-localized L-selectin for leukocyte tethering and rolling in model systems that mimic microvas-

L-selectin shedding is activated specifically within transmigrating pseudopods of monocytes to regulate cell polarity in vitro

“…Immunogold labeling of L-selectin showed that the majority of L-selectin was enriched on microvilli ( Fig. S2B), as has been shown previously for endogenous L-selectin on numerous primary leukocytes (17). Endogenous CaM was coprecipitated with L-selectin-GFP from whole-cell lysates, demonstrating interaction with known binding partners (Fig.…”

Comparative ultrastructure and expression of L-selectin on bovine αβ and γδ T cells

“…3A). Little or no L-selectin was detected on the rest of the plasmalemma of ␥␦ T cells, concurrent with the data previously reported for human leukocytes [20,21]. In contrast, WC1, a ␥␦ T cell subset marker, localized on the plasmalemma covering the ␥␦ T cell body but was absent on microvilli (Fig.…”

“…We also examined the expression of L-selectin on bovine lymphocytes to determine whether, as shown with human leukocytes [20,21], L-selectin is localized to the tips of microvilli. Lymphocytes were isolated as above, stained with DREG 56 (␣-L-selectin) or IL-A29 (␣-WC1) on ice, and visualized with goat anti-mouse IgG1 conjugated to 10 nM colloidal gold particles.…”

Event-Tracking Model of Adhesion Identifies Load-Bearing Bonds in Rolling Leukocytes

“…Human neutrophils, a type of leukocytes belonging to the innate immune system, have surface ruffles, called microvilli ( Figure 1A) [6,18], on which the sialomucin P-selectin glycoprotein ligand-1 (PSGL-1 or CD162) is expressed [33]. Most leukocyte rolling is mediated by PSGL-1 binding to P-selectin (CD62P) expressed on the inflammation-activated endothelium ( Figure 1B).…”

“…The load-bearing PSGL-1-P-selectin bonds are responsible for the jerky "stop-start" motion seen in rolling. There is extensive prior literature on computational [4,7,8,17,[21][22][23][24]43,45] and experimental studies [6,25,26,29,30,35,42,44] of leukocyte rolling. However, the load-bearing bonds-that is, bonds that determine the leukocyte's rolling behavior-have not been rigorously identified and investigated.…”