Lymphocytes In Efferent Lymph Research Articles

Estimating Residence Times of Lymphocytes in Ovine Lymph Nodes.

The ability of lymphocytes to recirculate between blood and secondary lymphoid tissues such as lymph nodes (LNs) and spleen is well established. Sheep have been used as an experimental system to study lymphocyte recirculation for decades and multiple studies document accumulation and loss of intravenously (i.v.) transferred lymphocytes in efferent lymph of various ovine LNs. Yet, surprisingly little work has been done to accurately quantify the dynamics of lymphocyte exit from the LNs and to estimate the average residence times of lymphocytes in ovine LNs. In this work we developed a series of mathematical models based on fundamental principles of lymphocyte recirculation in the body under non-inflammatory (resting) conditions. Our analysis suggested that in sheep, recirculating lymphocytes spend on average 3 h in the spleen and 20 h in skin or gut-draining LNs with a distribution of residence times in LNs following a skewed gamma (lognormal-like) distribution. Our mathematical models also suggested an explanation for a puzzling observation of the long-term persistence of i.v. transferred lymphocytes in the efferent lymph of the prescapular LN (pLN); the model predicted that this is a natural consequence of long-term persistence of the transferred lymphocytes in circulation. We also found that lymphocytes isolated from the skin-draining pLN have a 2-fold increased entry rate into the pLN as opposed to the mesenteric (gut-draining) LN (mLN). Likewise, lymphocytes from mLN had a 3-fold increased entry rate into the mLN as opposed to entry rate into pLN. In contrast, these cannulation data could not be explained by preferential retention of cells in LNs of their origin. Taken together, our work illustrates the power of mathematical modeling in describing the kinetics of lymphocyte migration in sheep and provides quantitative estimates of lymphocyte residence times in ovine LNs.

A method for tracking the migration of blood lymphocytes.

A method has been devised for labeling whole blood with the fluorescent dye fluorescein isothiocyanate (FITC) so the migration of blood lymphocytes can be studied in the sheep. Although lymphocytes can be purified from blood using density gradient media or elutriation it is difficult to obtain a large number of cells, because many cells are usually lost during the purification steps. It is desirable to label at least 10(8)-10(9) lymphocytes for lymphocyte tracking studies, because a smaller number is difficult to subsequently detect and quantitate in blood and lymph even using flow cytometry. Also, it is desirable to minimize the in vitro manipulation of lymphocytes, because dead or damaged lymphocytes will not recirculate. By labeling all the cellular components of a sample of whole blood rather than first purifying the lymphocytes we have been able to satisfy both of these criteria. Although labeled blood cells of all types are reinjected into the animal, the lymphocytes are easily distinguishable from other cells using a flow cytometer. In these studies between 2.4-12.4 x 10(8) lymphocytes were injected intravenously, and they were detectable in the blood and lymph for at least 10 days. The recovery of FITC-labeled (FITC+) lymphocytes in efferent lymph is comparable to that of lymphocytes labeled with other fluorescent or radioactive markers. The presence of labeled non-lymphoid cells in the animal makes this technique impractical for studies of lymphocyte localization within histologic sections. However, it is useful for studies in animals in which lymphatic vessels can be cannulated and the blood-to-lymph recirculation of labeled lymphocytes monitored, and it also may be applicable for studies in which lymphoid organ suspensions are analyzed using flow cytometry.

Pathways of lymph flow through superficial inguinal lymph nodes in the pig.

The pig lymph node has an unusual structure in that tissue containing lymph nodules generally occupies a central position. Our aim was to describe the lymphatic pathways through this node. We studied the structure of these pathways with light and electron microscopy, made casts of lymphatic vessels and sinuses with Microfil, and studied the distribution within the node of subcutaneously injected carbon particles. Most afferent lymphatics penetrate deeply within the node, where they give off several branches to peritrabecular sinuses that ramify through centrally located nodular tissue. However, where an afferent lymphatic enters the node there is a subcapsular sinus over an area of nodular tissue that occupies a conventional superficial position. Some lymph reaches this sinus from the central peritrabecular sinuses, but there can also be direct communications between this sinus and the afferent lymphatic. After flowing through sinuses in nodular tissue, lymph enters tissue that is analogous to medullary tissue in other species. This tissue is of two types, one consisting mainly of a diffuse network of reticular cells around spaces up to 10-12 microns across, and one that more closely resembles conventional medullary tissue. Lymph then flows to collecting ducts, which lack valves, and then to efferent lymphatics. Our findings do not support suggestions that a purely physical obstruction of lymphocytes in the lymph node accounts for the dearth of lymphocytes in efferent lymph of pigs.

Studies on the lymphocytes of sheep. II. Some properties of cells in various compartments of the recirculating lymphocyte pool.

The proportion of cells bearing surface immunoglobulin (Ig) and the normal lymphocyte transfer (NLT) reactivity of mononuclear cells obtained from the blood, afferent lymph, and efferent lymph from both somatic and intestinal nodes of sheep were measured. The proportion of Ig-positive lymphocytes was approximately 30% in blood and efferent lymph, but only about 10% in afferent lymph. The proportion of unlabeled lymphocytes (T cells) was about the same in all the preparations, as afferent lymph contains 15--20% macrophages. Despite this apparent uniformity in the T cell content, the NLT reactivity of efferent lymph was 3 to 5 times higher than that of blood or afferent lymph. No humoral factors could be detected by incubating cells in the various plasmas in vitro before injection. No inhibitory cells could be detected when mixtures of cell populations were used to initiate lesions. We suggest that lymphocytes in efferent lymph are more reactive than others because they are "groomed" by macrophages during their recirculatory passage through the substance of the node.