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Calcein: a novel marker for lymphocytes which enter lymph nodes
1Division of Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra.
Insights
Calcein dye uptake by lymphocytes identifies a subpopulation that preferentially homes to lymph nodes. This "dull" fluorescence phenotype correlates with lymph node homing receptor expression, suggesting membrane properties are key for HEV transmigration.
Area of Science:
- Immunology
- Cell Biology
- Biophysics
Background:
- Lymphocyte homing to lymphoid organs is crucial for immune surveillance.
- High endothelial venules (HEV) are specialized blood vessels facilitating lymphocyte entry into lymph nodes.
- Previous studies identified cell surface antigens as markers for lymphocyte-HEV binding.
Purpose of the Study:
- To investigate calcein dye uptake as a novel marker for lymphocyte homing.
- To characterize lymphocyte subpopulations based on calcein fluorescence intensity.
- To correlate calcein fluorescence with in vivo migration patterns and homing receptor expression.
Main Methods:
- Lymphocyte labeling with calcein and other fluorescein-based dyes.
- In vivo lymphocyte migration studies in mice.
- Analysis of lymphocyte fluorescence intensity and homing receptor (MEL-14) expression.
Main Results:
- Calcein differentially labels lymphocytes into "dull" and "bright" populations.
- "Dull" lymphocytes preferentially home to lymph nodes, including the popliteal lymph node (PLN).
- "Bright" lymphocytes are excluded from HEV-dependent entry but enter the spleen.
- A high percentage (76.5%) of "dull" lymphocytes express the PLN homing receptor MEL-14.
Conclusions:
- Calcein uptake serves as a marker for the lymph node-homing lymphocyte subpopulation.
- Differential calcein fluorescence reflects distinct lymphocyte migration behaviors.
- Calcein's utility suggests it may indicate membrane properties essential for HEV passage.
Abstract:
Previous studies have identified unique cell surface antigens which are associated with the specific binding of lymphocytes to high endothelial venules (HEV). Evidence is presented in this paper which demonstrates that uptake of the fluorescent dye calcein by lymphocytes represents an additional marker for the lymph node homing subpopulation of lymphocytes. Calcein exhibits a characteristic ability to label lymphocytes differentially into two distinct populations, based on fluorescence intensity, that does not occur with three other structurally related, fluorescein-based dyes. In vivo lymphocyte migration studies revealed that cells displaying the "dull" fluorescence phenotype, although entering all lymphoid organs examined, preferentially homed to the lymph nodes, particularly the popliteal lymph node (PLN). By contrast, lymphocytes displaying the "bright" phenotype were essentially excluded from entering lymphoid organs, where entry is HEV dependent, but were observed entering spleen, where entry is HEV independent. Furthermore, a high proportion (76.5%) of lymphocytes displaying the dull fluorescence phenotype expressed the PLN homing receptor MEL-14. Based on these observations it is suggested that calcein uptake may be a marker for general membrane properties, such as fluidity and plasticity, essential for the passage of lymphocytes through HEV.
