Intravital Microscopy of the Peripheral Lymph Node Microcirculation in Mice

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).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.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.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.