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Quantitation of L-selectin distribution on human leukocyte microvilli by immunogold labeling and electron microscopy
R E Bruehl1, T A Springer, D F Bainton
1Department of Pathology, University of California, San Francisco 94143, USA.
Abstract:
L-Selectin is a leukocyte cell adhesion receptor that contributes to neutrophil (PMN) rolling on activated endothelium at sites of inflammation 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 microvascular environments.
Insights
L-selectin, a key leukocyte receptor, is primarily located on the microvilli of neutrophils, monocytes, and lymphocytes. This distribution on leukocyte microvilli is crucial for understanding cell adhesion and rolling in inflammatory responses.
Area of Science:
- Cell Biology
- Immunology
- Biophysics
Background:
- L-selectin is a leukocyte cell adhesion receptor involved in neutrophil rolling and lymphocyte attachment.
- Previous studies indicated L-selectin localization on microvilli tips, but distribution and microvilli morphometry were unquantified.
- The distribution of L-selectin on various leukocytes and microvilli structure across subpopulations remained unexamined.
Purpose of the Study:
- To quantify L-selectin distribution on different leukocyte subpopulations.
- To examine the morphometry of microvilli on neutrophils, monocytes, and lymphocytes.
- To establish a foundation for studying the role of microvilli-localized L-selectin in leukocyte tethering and rolling.
Main Methods:
- Isolation of neutrophils (PMN) and mononuclear leukocytes from blood.
- Immunogold labeling of L-selectin on fixed cells.
- Transmission electron microscopy to analyze cell surface microstructure and L-selectin distribution via gold particle counting.
Main Results:
- L-selectin was predominantly found on microvilli: 78% on PMN, 72% on monocytes, and 71% on lymphocytes.
- Lymphocytes showed greater variance in L-selectin distribution compared to neutrophils and monocytes.
- Typical PMN and monocyte sections had 26 microvilli, while lymphocytes had 23.
Conclusions:
- Quantified L-selectin distribution and leukocyte surface topology provide a basis for further research.
- Understanding microvilli-localized L-selectin is essential for studying leukocyte adhesion and rolling.
- This study lays groundwork for investigating L-selectin's role in microvascular environments.