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Intravital microscopy of the peripheral lymph node microcirculation in mice
1Center for Blood Research, Harvard Medical School, Boston, Massachusetts, USA.
Insights
This study developed a new model to observe lymphocyte homing in mouse lymph nodes. The findings confirm L-selectin and PNAd are key for lymphocyte rolling in high endothelial venules.
Area of Science:
- Immunology
- Microcirculation Research
- Cell Biology
Background:
- Lymphocyte homing to lymph nodes is crucial for adaptive immunity.
- L-selectin (CD62L) and peripheral node addressin (PNAd) are critical for lymphocyte trafficking.
- Understanding these interactions requires advanced in vivo visualization techniques.
Purpose of the Study:
- To develop a novel intravital microscopy model for observing murine peripheral lymph node microcirculation.
- To characterize the functional role of L-selectin and PNAd in lymphocyte adhesion.
- To analyze the molecular mechanisms of lymphocyte homing to high endothelial venules (HEV).
Main Methods:
- Microsurgical dissection of murine subiliac lymph nodes.
- Intravital video microscopy to analyze microvascular architecture and hemodynamics.
- Fluorescently labeled L1-2 cells (transfected with or without L-selectin) were injected to study adhesion in venules, with and without blocking antibodies.
Main Results:
- The developed model enabled detailed analysis of leukocyte adhesion in lymph node microvessels.
- L-selectin-expressing cells exhibited rolling primarily in subcortical HEV and downstream venules.
- Rolling interactions were significantly reduced by antibodies targeting L-selectin and PNAd.
Conclusions:
- A new intravital microscopy model for visualizing leukocyte-lymph vessel interactions in murine lymph nodes was established.
- The study confirms L-selectin and PNAd as the primary receptor-ligand pair mediating lymphocyte rolling in HEV.
- This model offers a valuable tool for high-resolution analysis of events within living lymph nodes.
Objective:
The purpose of this study was to develop a model for microscopic in situ observation of the murine peripheral lymph node (LN) microcirculation and to characterize the function of the lymphocyte homing receptor L-selectin (CD62L) and the peripheral node addressin (PNAd). The latter is a high-affinity ligand for L-selectin in LN high endothelial venules (HEV).
Methods:
The subiliac (superficial inguinal) LN was microsurgically dissected in anesthetized adult mice. The nodal microvascular architecture and venular hemodynamics were characterized by bright field and epifluorescence video microscopy. L1-2 pre-B cells that were either mock transfected (L1-2Vector) or stably transfected to express human L-selectin (L1-2L-selectin) were labeled fluorescently and injected into a feeding artery. Cell adhesion in LN venules was studied in both the presence and absence of neutralizing monoclonal antibodies (MAb) to PNAd and L-selectin.
Results:
The preparation allowed a detailed analysis of hemodynamic parameters and leukocyte adhesion in LN microvessels. L1-2Vector cells did not interact with LN microvessels. In contrast, L1-2L-selectin cells rolled efficiently in venules but not in arterioles or capillaries. Rolling was most prominent in subcortical HEV (orders III to V) and was less frequent but consistently detectable in downstream medullary and hilus venules (orders I and II). Rolling interactions were abrogated by MAb DREG-56 to the lectin domain of L-selectin and were markedly reduced by the anti-PNAd MAb MECA-79.
Conclusions:
The present study develops a new intravital microscopy model for in vivo visualization of leukocyte interactions with microvessels in murine LN. The preparation permitted an analysis of biophysical and molecular mechanisms of leukocyte adhesion to high endothelial cells. The data support the concept that L-selectin and PNAd are the predominant receptor/ligand pair responsible for lymphocyte rolling in HEV. The model will be useful for high-resolution analysis of intra- and extravascular events in living LN.