Techniques to study lymphocyte migration

As in other processes in vivo, effective physiological study of the routes followed by migrating lymphocytes calls for minimal and innocuous intervention. First, use of lymphocytes from the blood or lymphatics, typically the thoracic duct, has the advantage that those lymphocytes are already in transit, in contrast to those extracted from lymphoid organs, which are a mixture of sessile and mobile lymphocytes. The cells must be labeled to be traced and this can be done by using isotopes, such as 51Cr, [-'HJ-cytidine or [JH]lysine to label all lymphocytes, or [3H|thymidine and [125I]uridine to label mainly lym-phoblasts since these isotopes are only incorporated in proliferating lymphocytes. Fluorescent dyes (e.g. fluorescein isothiocyanate, FITC) have proven valuable labels for tracing lymphocytes in vivo and in vitro. Used in combination with identification of surface markers, resting and activated lymphocyte subsets can be identified among migrating cells, both in suspension and also in tissue sections. In species in which congenic strains exist (e.g. mouse, rat, pig), lymphocytes can be transferred without any in vitro labeling procedure and the route of injected cells through lymphoid organs followed by identifying the cells with monoclonal antibodies. Thus, using adequate techniques the detailed routes of migration of lymphocyte subsets through lymphoid and non-lymphoid organs and their subcompartments are being identified.

Alternatively, in vitro techniques have proved very useful for the molecular study of lymphocyte adhesion to blood vessel endothelia. For example, lymphocyte binding to HEVs of lymphoid tissues can be analyzed on cryostat sections. HEV cells from lymph nodes in rodents can also be cultured in vitro, and the interaction between lymphocytes and these endothelial cells can be studied in more detail. Cultured human umbilical venous endothelial cells (HUVEC) are often used to study the mechanisms involved in lymphocyte binding. However, it has to be considered that this endothelium might differ in major aspects from the endothelia in lymphoid organs. A major advantage was the establishment of in vivo videomicroscopy, allowing the analysis of migrating lymphocytes under physiological shear forces.

In all the different techniques used to study lymphocyte traffic, the extent to which the methodology may have altered the physiological processes has to be considered and compared critically.

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