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Analysis of L-selectin-mediated cellular interactions under flow conditions
1Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, 142-8501, Tokyo, Japan, h-kawashima@hoshi.ac.jp.
Insights
Two methods detect L-selectin-mediated lymphocyte homing to lymph nodes under flow. These assays assess leukocyte interactions with high endothelial venules (HEVs) and can test agents affecting cell recruitment.
Area of Science:
- Immunology
- Cell Biology
- Physiology
Background:
- Lymphocyte homing is crucial for immune surveillance and is mediated by L-selectin binding to ligands on high endothelial venules (HEVs).
- Understanding these interactions under physiological flow conditions is essential for studying immune cell trafficking.
Purpose of the Study:
- To describe two distinct experimental methods for analyzing L-selectin-mediated lymphocyte-HEV interactions under shear stress.
- To provide tools for evaluating agents that modulate lymphocyte homing and recruitment.
Main Methods:
- A modified Stamper-Woodruff assay using fluorescently labeled leukocytes and frozen lymph node sections under shear stress.
- A parallel flow chamber assay to observe leukocyte rolling on L-selectin ligand-expressing cells under varying shear stress levels.
Main Results:
- The modified Stamper-Woodruff assay allows clear visualization of leukocyte binding to HEVs via fluorescence microscopy.
- The parallel flow chamber assay enables quantitative assessment of leukocyte adhesion dynamics under simulated physiological flow.
Conclusions:
- These described methods offer robust approaches to study L-selectin-dependent lymphocyte homing.
- The assays are applicable for screening compounds or biological agents that influence lymphocyte recruitment to lymph nodes.
Abstract:
Lymphocyte homing is mediated by a specific interaction between L-selectin expressed on lymphocytes and its ligands expressed on high endothelial venules (HEVs) in lymph nodes under physiological flow conditions. In this chapter, two methods for detecting L-selectin-mediated cellular interactions under shear stress mimicking physiological flow conditions are described. First, a modified Stamper-Woodruff cell-binding assay using leukocytes labeled with a fluorescent orange dye, CMTMR, is introduced. In this method, leukocytes are allowed to bind to frozen lymph node sections under shear stress and their binding to HEVs can be clearly visualized by fluorescence microscopy. Second, a parallel flow chamber assay is described. In this assay, leukocytes are allowed to roll on L-selectin ligand-expressing cells under various levels of shear stress and their adhesive interactions are recorded by a video camera equipped with an inverted microscope. These methods can be applied to determine the effects of various agents that might affect L-selectin-mediated lymphocyte homing and recruitment.
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