Lymphocyte migration in syngeneic lymph node implants

Lymph nodes implanted subcutaneously to syngeneic recipients were shown to regenerate after mass cell destruction. Regenerated lymphoid tissue has a resemblance to the cortical zone of intact lymph nodes. Microenvironment of regenerated lymphoid tissue provides homing of lymphocytes. However, migration of 51Cr-labelled lymphocytes to implants declined drastically, as compared to lymphocyte migration to intact lymph nodes. Attenuation of proliferative activity and the data of morphological analysis indicate a more prolonged retention of lymphocytes in implanted lymph nodes. The results obtained could be attributable to only partial recovery of sinus and vessel systems regulating the inflow and outflow of lymphocytes in lymph nodes.     

Modification of lymphocyte migration by mannans and phosphomannans. Different carbohydrate structures control entry of lymphocytes into spleen and lymph nodes

Previous in vitro studies suggest that recognition of phosphomannosyl structures by lymphocytes plays a central role in the binding of lymphocytes to high endothelial venules. However, the physiologic relevance of phosphomannosyl recognition in in vivo lymphocyte migration has not been established. This paper describes experiments that examined this question. It was demonstrated that the phosphomannan monoester core (PPME) from Pichia holstii, a potent inhibitor of peripheral node high endothelial venule interactions in vitro, was a very effective inhibitor of in vivo lymphocyte migration, as little as 39 micrograms/mouse significantly inhibiting popliteal lymph node entry. Furthermore, PPME exhibited a similar hierarchy of inhibition in vivo as previously reported in vitro, most effectively inhibiting entry of lymphocytes into popliteal lymph node, somewhat less effectively inhibiting mesenteric lymph node entry and being a relatively poor inhibitor of Peyer's patch entry. Additionally, PPME inhibited splenic entry of lymphocytes, and inhibition of lymphoid organ entry was accompanied by a substantial leukocytosis. Two additional mannose-containing compounds were found to modify lymphocyte migration, namely a well defined mannose containing pentasaccharide (PENT) with terminal mannose-6-phosphate (M6P) and an unphosphorylated yeast mannan. Both PENT and mannan induced leukocytosis and were particularly effective at inhibiting splenic entry of lymphocytes. In fact, detailed dose-response curves indicated that mannan was a much more potent inhibitor of splenic entry than PPME or PENT, whereas in lymph nodes PPME was the most effective inhibitor. Pretreatment of lymphocytes before injection with either PPME or mannan demonstrated that PPME could act at the lymphocyte level, whereas mannan probably acted at some other site. Collectively, these data suggest that different carbohydrate structures are involved in the entry of lymphocytes into different lymphoid organs, with mannose recognition playing an important role in splenic entry and recognition of M6P-like structures controlling lymph node entry. In contrast, it was found that mannose-and M6P-containing structures, unlike sulfated polysaccharides such as fucoidan, did not affect the subsequent positioning of lymphocytes within lymphoid organs.     

Abnormal migration of T lymphocyte clones

Several in vitro T cell clones were markedly deficient in their ability to home to peripheral lymphoid tissue. This was found for an alloreactive noncytolytic clone, a soluble antigen- (KLH)specific line, and cytotoxic clones specific for allogeneic cells and for Abelson virus-induced lymphoma cells. This abnormal circulation pattern was probably caused by the lack of the receptors of the lymphocytes for high endothelial venules (HEV), as implied by the lack of binding of these T cells to HEV in frozen sections of mouse lymph node and Peyer's patches. The loss of surface receptors that are necessary for normal lymphocyte migration may thereby alter the in vivo function of adoptively transferred T cells.     

Lymphocyte migration in the body in conditions of internal irradiation. 2. Disorders of lymphocyte microenvironmental factors in organs

At later times after the injection of selenium-75-selenium methionine to CBA mice the inhibition of migration of intravenously injected normal 51Cr lymphocytes to the lymph nodes and liver lasted for a long time. On the contrary, no changes in the normal lymphocyte migration within the animal body were observed after total-body gamma-irradiation with the dose of 4 Gy. The data obtained are indicative of the radiation-induced disturbance of the microenvironment factors, which provide migration of lymphocytes within the body, during the long-term exposure to radiation emitted by the incorporated source.     

Effects of fluorescent and nonfluorescent tracing methods on lymphocyte migration in vivo.

BACKGROUND: The use of fluorescent dyes to monitor in vivo cellular migration and proliferation has greatly expanded, but little is known about their potential influence on cell migration. METHODS: Adoptive transfer studies of lymphocytes labeled with various dyes were performed, and their in vivo homing was compared with that of coinjected unlabeled control cells. In addition, in vitro migration and binding studies were performed to analyze the various steps of transmigration separately. RESULTS: These data showed that the intracellular fluorescent dyes calcein acetoxymethyl ester, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester, 5-chloromethylfluorescein diacetate, 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, and fluorescein isothiocyanate affect in vivo homing of especially B lymphocytes to lymphoid organs, without any direct effect on in vitro chemotactic or adhesive activity. The only label that did not affect migration was the extracellular and nonfluorescent molecule biotin, provided that the labeling was performed at room temperature. Interestingly, by using the highly versatile congenic Ly5.1-Ly5.2 system, we also demonstrated intrinsic differences in lymphocyte migration based on allelic differences. CONCLUSIONS: Our data showed that fluorescent labeling of lymphocytes has a severe effect on their homing capacity in vivo. Labeling of cells with biotin appeared to be a good alternative for this purpose; however, if direct fluorescence is required, the negative effects on cell migration should be considered.     

Dendritic cell migration and lymphocyte homing imprinting

For an effective adaptive immune response to occur, dendritic cells (DC), which are the most efficient antigen-presenting cells, must be able to sample the peripheral microenvironment and migrate towards secondary lymphoid organs (SLO) where they activate naive lymphocytes. Upon activation, lymphocytes proliferate and acquire the capacity to migrate to extralymphoid compartments. Although the molecular mechanisms controlling lymphocyte homing to lymphoid and to some extralymphoid tissues have been described in significant detail, it is much less clear how DC migration is controlled. Do DC obey similar adhesion cues that lymphocytes do, or do they have their own "zip codes"? This is relevant from a therapeutic standpoint because effective DC-based vaccines should be able to reach the appropriate tissues in order to generate protective immune responses. Here, we discuss some of the mechanisms used by DC to reach their target tissues. Once DC arrive at their destination, they are exposed to the tissue microenvironment, which likely modulates their functional properties in a tissue-specific fashion. This local DC "education" is probably responsible among other things; for the acquisition of tissue-specific homing imprinting capacity by which DC instruct lymphocytes to migrate to specific tissues. Finally, we discuss how dysregulation of these signals may play a key role in disease.     

In vitro studies on transplantation immunity. II. The migration inhibition in homograft reactions in guinea pigs

The migration of peritoneal exudate cells from guinea pigs exhibiting transplantation immunity is inhibited in the presence of donor antigens. This inhibition of migration is demonstrable whether the donor transplantation antigens are presented in the form of viable cells (peritoneal exudate cells) or as particulate subcellular antigens (spleen microsomes). A greater degree of inhibition was observed when transplantation immunity was induced with lymphoid cells in Freud's adjuvant compared to sensitization with orthotopic skin grafts. There was no inhibition of migration in mixtures of normal allogeneic cells or when peritoneal cells from guinea pigs exhibiting tuberculin hypersensitivity were mixed with similar cells from normal animals. Finally, supernatants from cultures of sensitive lymphocytes plus donor antigens inhibited the migration of normal peritoneal cells indicating the presence of migration inhibitory factor (MIF) activity.     

Neuronal repellent Slit2 inhibits dendritic cell migration and the development of immune responses

One of the essential functions of dendritic cells is to take up Ags in peripheral tissues and migrate into secondary lymphoid organs to present Ags to lymphocytes for the induction of immune responses. Although many studies have demonstrated that the migration of dendritic cells is closely associated with the development of immune responses, little is known about factors that inhibit dendritic cell migration and control the extent of immune responses to Ag stimulation. We show that Slit2, a neuronal repellent factor, is up-regulated in the skin by allergen sensitization and down-regulates the migration of Langerhans cells. The effect is mediated by direct interaction of Slit2 with cells that express a Slit-specific receptor, Robo1. Slit2-mediated inhibition of Langerhans cell migration results in suppression of contact hypersensitivity responses. These findings provide insights into a novel mechanism by which Slit2 functions as an anti-inflammatory factor for the initiation of immune responses.     

The effect of ionizing radiation on lipid metabolism in lymphoid cells

Lipid metabolism was studied in lymphoid tissues of rats after whole body irradiation with doses producing damage of different degrees to lymphoid cells (4-10 Gy). The content of free cholesterol, cholesterol esters, and total phospholipids was determined in peripheral blood lymphocytes and thymocytes 1-2 h after exposure. Simultaneously, the rate of in vitro incorporation of 2 14C-acetate into total lipids, phospholipids, and cholesterol of lymphoid cells was estimated. It was shown that exposure of rats to ionizing radiation caused activation of lipogenesis. Cholesterol synthesis was activated after a dose of 4 Gy and decreased with increasing dose.     

The production of heterologous antibodies to the human lymphakine: leukocyte migration inhibitory factor (LIF).

Human leukocyte migration inhibitory factor (LIF) produced by concanavalin A-stimulated lymphocytes was partially purified by Sephadex G-100 chromatography and immunosorption of protein contaminants. This material was injected into two rabbits, and the IgG-IgA fractions of the resulting antisera (anti-LIF) neutralized LIF induced by antigen (PPD tuberculin) with as equal efficiency as that of LIF induced by mitogen. Anti-LIF activity was neither removed by absorption with control supernatant or normal human serum nor was it suppressed by absorption with lymphocytes or lymphoblasts. On the other hand, antibodies against human lymphoid cells (ALG) did not reduce LIF activity, indicating the difference between anti-LIF and classical ALG. In support of this, anti-LIF, in contrast to ALG, was not cytotoxic to lymphocytes, and it did not inhibit spontaneous T-rosette formation with sheep erythrocytes. In crossed immunoelectrophoresis using a conventional proteinstaining technique, only three precipitates appeared. None of these contained LIF. However, a protein migrating in the prealbumin region appeared to be specific for lymphocyte stimulation. The nature and significance of this product is unknown.     

Inhibition of migration of tumor cells in vitro by lymphokine-containing supernatants.

Lymphokine-containing supernatants derived from seven different human lymphoid cell lines and lymphokine-containing supernatants from concanavalin A-stimulated murine lymphocytes were found to be capable of reversibly inhibiting the migration of tumor cells in vitro. The tumor cell lines used in these studies were the P815 mastocytoma, Ehrlich ascites, Walker carcinosarcoma, Hepatoma 129, and Sarcoma 37. Preliminary physiochemical evidence suggests that the mediator, here termed TMIF, is distinct from MIF. In any case, these results suggest the possibility that lymphokines other than lymphotoxin or macrophage-activating factors may play a role in tumor immunity.     

Haptotactic activity of fibronectin on lymphocyte migration in vitro

In addition to mediating cell adhesion, fibronectin (FN) also affects the migration of different cell types. However, the role of FN in lymphocyte migration is unclear. In this study, we examined the effects of FN on the in vitro migration of lymphocytes. Using the checkerboard analysis in a blind-well microchemotaxis assay, soluble FN was determined to have neither a chemotactic nor chemokinetic effect on spleen or thymus lymphocytes. However, when the nitrocellulose filter was coated unidirectionally with FN, the migration of both spleen and thymus lymphocytes into the filter was enhanced, indicating that FN is haptotactic for lymphocytes. When the filter was coated bidirectionally, no enhancement in migration was observed, indicating that FN is not haptokinetic for lymphocytes. When the FN cell-binding domain and the heparin-binding domain were tested, the cell-binding domain was haptotactic for both spleen and thymus lymphocytes, whereas the heparin-binding domain was only haptotactic for spleen lymphocytes. Because the heparin-binding domain can mediate strong adhesion of thymus lymphocytes, the lack of haptotactic activity is likely to be the result of excessive binding that prevents cell motility.     

Splenic lymphocytopoiesis and migration pattern of splenic lymphocytes

The spleens of young pigs were selectively labeled with tritiated thymidine ([³H]-TdR) and the relative and absolute numbers of labeled lymphocytes found 24 hr later in different lymphoid and nonlymphoid organs were determined autoradiographically. It was deduced that about 4.6 × 10⁹ lymphocytes (that is, about 15% of all splenic lymphocytes) are produced by the spleen per day and about 17% of the newly formed lymphocytes leave the spleen within the first day of labeling. Spleen-derived lymphocytes could be found in relatively high numbers in the lymph nodes, blood, gut-associated lymphoid tissues, and, surprisingly, in the bone marrow, whereas the concentration in the thymus was very low. In a second series, pigs were labeled with [³H]TdR and only the spleen was excluded from labeling. The labeling index of splenic small lymphocytes was about 10% 1 day later, indicating a high rate of influx of newly formed lymphocytes into the pig spleen. The spleen of the young pig is an important lymphocytopoietic organ and exports and imports newly formed lymphocytes at high rates.     

Modelling of peripheral lymphocyte migration: system identification approach

This is the first application of the prediction error method (PEM) of system identification to modelling lymphocyte migration through peripheral lymphoid tissue. The PEM was applied to the emergence of labelled lymphocytes from the efferent lymphatic of a lymph node following their intravenous administration. Advantages of PEM included the capacity to calculate the response to a unit impulse stimulus, unavailable to direct observation, and to allow for the return to the node of labelled cells that had already recirculated once. Calculation of the system delay (time between introduction of cells into the blood and their first appearance in lymph) indicated 4.67 +/- 1.05 h for the total lymphocyte population. The peak in efferent lymph occurred at 11.91 +/- 4.68 h, much earlier than previous reports, which were affected by cells that had already recirculated. While 75% of labelled cells had emerged in efferent lymph by 20.77 +/- 5.62 h, 86.38 +/- 29.44 h was required for 100% emergence. The considerable heterogeneity in migratory behaviour is likely to reflect frequency and duration of binding of lymphocytes by dendritic cells in paracortical cord corridors. It is proposed that differences in the speed with which lymphocytes pass along corridors depend on their functional status, in particular whether they are na?ve or memory cells.     

Central role of paxillin phosphorylation in regulation of LFA-1 integrins activity and lymphocyte migration

Coordinated changes of actin cytoskeleton and cell adhesion accompany maturation of lymphoid cells, their migration through lymphoid organs and to sites of inflammation, as well as metastasis of transformed cells. Here we discuss the central role of the actin-regulating adaptor protein, paxillin, during lymphocyte transition from a polarized, motile cell phenotype with partially active LFA-1 integrins to a round and immobile one with fully active LFA-1. In Baf3 murine pro-B lymphocytes, the former phenotype is induced by IL-3 that stimulates a FAK-mediated phosphorylation of paxillin at tyrosines (Y) 31 and 118 and a consequent Rac1 activation. Rearrangements of actin cytoskeleton that lead to the cell''s acquisition of a spherical shape and LFA-1 activation are achieved upon activation of PKC-δ that binds and directly phosphorylates paxillin at threonine (T) 538 with consequent RhoA activation. This is accompanied by dephosphorylation of paxillin Y31/118 and by Rac1 inactivation. We propose a model of signaling cascades that reflects the interplay between the IL-3- and PKC-δ-mediated pathways.Key words: lymphocytes, paxillin, actin, Rac1, RhoA, LFA-1, PKC-δ     

CD73-generated adenosine restricts lymphocyte migration into draining lymph nodes

After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naive T cells with Ag-loaded dendritic cells. In this study, we show that CD73 (ecto-5'-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73(-/-) mice have normal sized lymphoid organs in the steady state, but approximately 1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared with wild-type mice 24 h after LPS administration. Migration rates of cd73(+/+) and cd73(-/-) lymphocytes into lymph nodes of wild-type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A(2B) receptor is a likely target of CD73-generated adenosine, because it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up-regulated by TNF-alpha. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73(-/-) mice is largely normalized by pretreatment with the selective A(2B) receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.     

Detection of heterogenicity of the lymphoid populations by measuring the diameter of the cells and cell nuclei]

Some parameters of distribution according to the lymphoid cell and its nuclei size in the peripheral blood, bone marrow, thymus and spleen of healthy rats were studied. A comparative assay revealed population homogeneity for the thymus and bone marrow lymphocytes as well as their mean diameter differences. Mixing of these cell types markedly changed the distribution parameters of newly formed population, as shown on the model of the bone marrow lymphoid reaction caused by migration of thymic lymphocytes after 5-fluorouracil use. Preliminary thymectomy excluded migration and homogeneity of the bone marrow lymphocyte size remained unchanged.     

Pathways of lymphocyte migration within the periarterial lymphoid sheath of rat spleen

Summary Male Wistar rats were injected intravenously with 5-(³H)uridine-labeled lymphocytes isolated from lymph nodes of syngeneic donors and enriched in T cells. After short periods of time (3 to 120 min after injection), labeled lymphocytes were localized in spleen compartments using autoradiography to identify routes of lymphocyte movement from blood into splenic parenchyma and to follow migration pathways of recirculating lymphocytes within the periarterial lymphoid sheath (PALS). Topographical analysis of labeled lymphocytes was performed in specific planes of PALS characterized by the diameter of the arterial vessel and termed PALS large, PALS medium, and PALS small (PALS L, PALS M, PALS S, respectively). Attention was also paid to accumulations of labeled lymphocytes close to the arterial vessel wall. Initially, labeled lymphocytes were localized in PALS S and PALS M near the terminal branching of arterial vessels and in the marginal zone (MZ). We conclude that lymphocytes emigrate from blood into splenic parenchyma within two white pulp compartments: in MZ, and directly within PALS through the wall of capillary vessels. The sequential accumulation of labeled cells near arterial vessels of increasing diameter suggests that the recirculating pool of lymphocytes migrates into the central part of PALS L by two routes: from MZ, and along arterial vessels from PALS S and PALS M.R.B. was a fellow of the Alexander von Humboldt-Stiftung, on leave from the Department of Histology and Embryology, Institut of Biostructure, Academy of Medicine, ul. Swiecickiego 6, PL-60-781 Pozna, Poland.