The kinetics of antigen-reactive cells during lymphocyte recruitment

Lymphocyte recruitment, the increased traffic of lymphocytes from blood to lymph which occurs within antigenically stimulated lymph nodes, was monitored in the efferent lymph of single lymph nodes in sheep after immunization with allogeneic lymphocytes or purified protein derivative. Specific antigen-reactive cells were assayed by their ability to proliferate in vitro in the presence of the priming antigen. During lymphocyte recruitment such cells were no longer detected in the efferent lymph draining either the immunized node or a nonstimulated node remote from the region of antigen administration. These results probably reflect the selective removal of specific lymphocytes from the recirculating pool. Alternatively, the findings could involve a state of specific unresponsiveness of the cells.     

Directional Migration of Recirculating Lymphocytes through Lymph Nodes via Random Walks

Naive T lymphocytes exhibit extensive antigen-independent recirculation between blood and lymph nodes, where they may encounter dendritic cells carrying cognate antigen. We examine how long different T cells may spend in an individual lymph node by examining data from long term cannulation of blood and efferent lymphatics of a single lymph node in the sheep. We determine empirically the distribution of transit times of migrating T cells by applying the Least Absolute Shrinkage & Selection Operator () or regularised to fit experimental data describing the proportion of labelled infused cells in blood and efferent lymphatics over time. The optimal inferred solution reveals a distribution with high variance and strong skew. The mode transit time is typically between 10 and 20 hours, but a significant number of cells spend more than 70 hours before exiting. We complement the empirical machine learning based approach by modelling lymphocyte passage through the lymph node . On the basis of previous two photon analysis of lymphocyte movement, we optimised distributions which describe the transit times (first passage times) of discrete one dimensional and continuous (Brownian) three dimensional random walks with drift. The optimal fit is obtained when drift is small, i.e. the ratio of probabilities of migrating forward and backward within the node is close to one. These distributions are qualitatively similar to the inferred empirical distribution, with high variance and strong skew. In contrast, an optimised normal distribution of transit times (symmetrical around mean) fitted the data poorly. The results demonstrate that the rapid recirculation of lymphocytes observed at a macro level is compatible with predominantly randomised movement within lymph nodes, and significant probabilities of long transit times. We discuss how this pattern of migration may contribute to facilitating interactions between low frequency T cells and antigen presenting cells carrying cognate antigen.     

Stochastic regulation of cell migration from the efferent lymph to oxazolone-stimulated skin

The systemic immune response is a dynamic process involving the trafficking of lymphocytes from the Ag-stimulated lymph node to the peripheral tissue. Studies in sheep have demonstrated several phases of cell output in the efferent lymph after Ag stimulation. When skin contact sensitizers are used as Ag, the efferent lymph cell output peaks approximately 96 h after Ag stimulation and is temporally associated with the recruitment of cells into the skin. To investigate the relative contribution of this high-output phase of efferent lymphocytes to lymphocytic inflammation in the skin, we used a common contact sensitizer 2-phenyl-4-ethoxymethylene-5-oxazolone (oxazolone) to stimulate the skin and draining prescapular lymph node of adult sheep. The efferent lymph ducts draining the Ag-stimulated and contralateral control lymph nodes were cannulated throughout the experimental period. The lymphocytes leaving the lymph nodes during the 72-h period before maximum infiltration were differentially labeled with fluorescent tracers, reinjected into the arterial circulation, and tracked to the site of Ag stimulation. Quantitative tissue cytometry of the skin at the conclusion of the injection period (96 h after Ag stimulation) demonstrated more migratory cells derived from the Ag-stimulated lymph node than the contralateral control (median 18.5 vs 15.5 per field; p < 0.05). However, when corrected for total cell output of the lymph node, the Ag-stimulated migratory cells were 3.8-fold more prevalent in the skin than the contralateral control cells. These results suggest that the in situ immune response generally mirrors the frequency of recruitable lymphocytes in the peripheral blood.     

Lymphocyte recruitment in delayed-type hypersensitivity. The role of IFN-gamma

Lymphocytes are recruited out of the blood into delayed-type hypersensitivity (DTH) reactions, but the factors controlling their migration are poorly understood. Our previous studies have shown that IFN-alpha/beta, its inducers, and T cell lymphokines can induce lymphocyte migration into the skin after intradermal injection. The present studies were designed to determine the effect of rIFN-gamma, IL-1, and anti-IFN-gamma on lymphocyte recruitment into DTH. Small peritoneal exudate lymphocytes, which preferentially migrate to inflammatory sites, were labelled with 111In and injected i.v. into rats. The intradermal injection of IFN-gamma stimulated the migration of these lymphocytes into the skin. IL-1 induced very little migration by itself, but enhanced the effect of IFN-gamma. Kinetic analysis demonstrated that the migration of lymphocytes to IFN-gamma was rapid, with a peak at 6 h, whereas migration into a DTH reaction was minimal for the first 8 h and reached a peak 24 h after intradermal injection. Polyclonal rabbit anti-IFN-gamma anti-serum, and a Mab to IFN-gamma, DB-2, could almost completely block lymphocyte migration induced by IFN-gamma. Furthermore, DB-2 inhibited lymphocyte recruitment into DTH reactions by 50 to 90%. This Mab did not affect migration in response to IFN-alpha/beta, although it partially inhibited the response to polyI:C. The effect of IFN-gamma on lymphocyte recruitment was not specific for small peritoneal exudate lymphocytes, because both spleen T cells and lymph node cells migrated in response to IFN-gamma and DB-2 inhibited the recruitment of splenic T cells to DTH. Thus, IFN-gamma is a potent stimulator of lymphocyte migration into the skin and a major mediator of lymphocyte recruitment into DTH.     

The unique ultrastructure of high-endothelial venules in inguinal lymph nodes of the pig

Lymph nodes in pigs are unique in their inverted structure, with the medulla in the periphery and the cortex in central areas. Furthermore, in this species most migrating lymphocytes do not use the classical route via efferent lymphatics to leave the lymph node. High-endothelial venules (HEV) are the entry sites for lymphocytes and in pigs probably also the exit site for recirculating lymphocytes. Therefore, the blood vessels and especially the HEV of the pig superficial inguinal lymph node were investigated as to whether morphological peculiarities could be found in the vascular system, using vascular casting, transmission- and scanning electron microscopy. A thin layer of capillary network surrounded the periphery of the lymph node and HEV branched acutely. The endothelial cells of HEV possessed well developed cytoplasmic organelles, interdigitated with each other, and demonstrated local cell-cell contacts. There were unusual cells bridging the adluminal wall of HEV. These cells were called intravascular bridging cells. They were characterized by an often invaginated nucleus, few pinocytotic vesicles, many microvilli on the surface, wide, flat, cytoplasmic processes like a pseudopod, Weibel-Palade bodies and local cell-cell contacts with endothelial cells. The pseudopod-like processes ramified over the endothelial junctions and covered lymphocytes. Lymphocytes were seen in different phases of migration between endothelial cells and in the intercellular junctions. The previous functional studies on the peculiar route of lymphocyte recirculation in pig lymph nodes are extended by these morphological data, showing a unique structure of HEV in pigs.     

Response of the regional lymphatic system of the sheep to acute stress and adrenaline.

An acute pain stimulus resulted in elevated lymph flow and output of cells from the popliteal lymph node of the sheep in the first 15 min after the stress. Efferent lymph flow increased by an average of 93% above the mean resting flow and cell output rose by an average of 170% during this period, but by 30 min after the stress, values for both lymph flow and cell output had returned to normal. The cell content of the efferent lymph was significantly higher in the first 15 min after the acute stress and it is suggested that there is a sizeable pool of lymphocytes within the resting popliteal node which can be mobilized into the lymph by an acute stress. A single intravenous injection of 1 mg adrenaline the efferent lymph flow in all the sheep examined but gave rise to an increased cell output in only 50% of the sheep. This indicated that there may be other factors, possibly hormonal, involved in the movement of the pool of lymphocytes out of the regional lymph node following acute stress. Both acute pain stress and adrenaline resulted in an increased afferent popliteal lymph flow and output of cells from the regional tissues in the first 15 min after administration. The results are suggestive of a small pool of lymphocytes in the regional tissues which may be readily mobilized by either acute stress or adrenaline. Part of the increases in efferent and afferent lymph flow observed following acute stress and adrenaline appeared to be due to an increased lymph formation, presumably as a result of an increased capillary pressure. Nevertheless, it is considered that the greater part of the increased flow of lymph from both regions resulted from an accelerated movement of performed lymph.     

High endothelial venule morphology and function are inducible in germ-free mice: a possible role for interferon-gamma

Cytokines may facilitate lymphocyte traffic by modulating HEV structure and lymphocyte binding function in accordance with local tissue requirements. This study investigated whether the morphology of HEVs and their lymphocyte binding ligand are altered following antigenic challenge and evaluated the role of IFN-gamma in the induction of such changes. The morphology and lymphocyte binding function of mesenteric LN HEVs of GFM exposed to environmental pathogens were compared to those from GFM and conventional mice. Lymph nodes from all mice had microscopically identifiable HEVs. The morphology of HEVs from GFM was not uniform; many HEVs contained flat endothelial cells with sparse cytoplasm and prominent interendothelial gaps. The number of lymphocytes within the lumen and the HEV wall was low. In contrast, HEVs from GFME and conventional mice were characterized by cuboidal endothelial cells with plentiful cytoplasm and large numbers of lymphocytes in the vessel wall and lumen. There was no delineation of interendothelial cell borders. Lymphocyte binding to HEVs of lymph node sections from GFM was reduced (mean +/- SEM: 1.08 +/- 0.15) compared to that of conventional mice (1.91 +/- 0.20), P less than 0.003. GFME had augmented lymphocyte binding (2.23 +/- 0.26) to levels comparable with those of conventional mice. GFM injected intraperitoneally with IFN-gamma, IFN-alpha beta, or diluent resulted in minor changes in HEV morphology. By contrast, lymphocyte binding to HEV of GFM was more than doubled by the injection of IFN-gamma (1.95 +/- 0.25), P less than 0.01, but not IFN-alpha beta (0.54 +/- 0.07) or the relevant diluent controls (0.89 +/- 0.11, 0.56 +/- 0.06, respectively). It appears that the HEV binding ligand is inducible, and its expression is regulated by at least one immunomodulator, IFN-gamma. Although short-term exposure of HEVs to IFN-gamma influenced HEV function it caused only minor changes in morphology.     

Lymph node, spleen and peripheral blood lymphocytes as stimulators of alloreactivity

Before and after kidney transplantations, in vitro tests that measure the level of reactivity between donor and recipient lymphocytes are performed for better organ selection and as indicator of possible organ rejection. In these tests, donor's and recipient's lymphocytes are stimulated for proliferation, which intensity is measured and accordingly organ recipient reactivity towards graft is determined. Lymph node, spleen and peripheral blood lymphocytes are used for those purposes. For better interpretation of these in vitro tests it should be important to determine mitogenic ability of lymphocytes of different origin and to choose the most adequate cells. To compare mitogenic ability of deceased donor lymph node, spleen and peripheral blood lymphocytes one-way mixed lymphocyte culture (MLC) was used. As stimulators irradiated lymphocytes from spleen, lymph node and peripheral blood samples of 12 deceased donors were used while as responders lymphocytes from peripheral blood of healthy individuals, chosen according HLA-DRB1 alleles (stimulators and responders were HLA-DRB1 identical, semi-identical or different), were used. Spleen lymphocyte activity was the best with different cells and the weakest with identical cells. Impact of polyclonal mitogens (PHA - phytohemagglutinin, Con A - concanavalin A and PWM - pokeweed mitogen) on lymphocyte proliferation was tested on lymphocytes from spleen and lymph node of deceased donors. Results obtained in culture in vitro showed that spleen cells had exerted the best mitogenic potential and PHA had the greatest impact upon lymphocyte proliferation. This investigation is of importance for establishing the best model to reflect in vivo situation in transplanted patient.     

Plasmid DNA induces increased lymphocyte trafficking: a specific role for CpG motifs

Bacterial DNA, primarily through immunostimulatory cytosine-guanine (CpG) motifs, induces the secretion of cytokines and activates a variety of effector cells. We investigated the possibility that CpG motifs might also modulate immunosurveillance by altering cell trafficking through a regional lymph node. Intradermal injection of plasmid DNA induced rapid and prolonged increases in the number of lymphocytes collected in efferent lymph. This effect on cell trafficking was not dependent on the expression of an encoded reporter gene but varied with plasmid construct and required a circular form. Injection of synthetic oligodeoxyribonucleotides containing CpG motifs did not alter lymphocyte trafficking but CpG-enhanced plasmid induced a dose-dependent increase in cell trafficking. Phenotypic analyses revealed that the increase in cell trafficking involved all lymphocyte subpopulations and represented a mass movement of cells. These observations reveal that bacterial DNA, through immunostimulatory CpG motifs, alters immunosurveillance by increasing cell recruitment to a regional lymph node.     

The production of T-cell growth factor (TCGF) in vivo in sheep

Lymph and supernatants derived from efferent lymphocytes leaving the popliteal lymph nodes of sheep responding to human red cells or dinitrophenylated bovine serum albumin were examined for the presence of T-cell growth factor (TCGF). Efferent cells from normal sheep, but not from antigen-stimulated sheep, were found to release low levels of TCGF when incubated in medium for 12 hr in the absence of any exogenous stimulus. High levels of TCGF were found in normal lymph and also in immune lymph collected from sheep during the first 6 hr of immune responses. There were no detectable levels of TCGF in lymph collected later in the response. The lymphokine appeared to be a single molecular species of 10,000–20,000 molecular weight as assessed by exclusion chromatography. Efferent cells expressing receptors for TCGF were found in efferent lymph during the first 12 hr of the response. The results demonstrate for the first time that TCGF is produced in vivo and that asynchrony exists between TCGF production and expression of receptors for TCGF on efferent cells released by the stimulated node. Based on the known kinetics of previously reported synergistic factors, mitogenic factors, and T-cell-replacing factors in sheep efferent lymph and their physical characteristics it was concluded that the TCGF detected in lymph is distinct from these factors.     

'Morphology of immune lymphocytes in homograft lymph'

Uropod-bearing lymphocytes (UBLs) resembling cytotoxic cells found in vitro were identified in lymph collected from rabbit hind-limbs bearing homografts. UBLs were found in lymph leaving the regional lymph node (efferent), both before and after rejection, but not in that draining the homograft (afferent) even though they were present in the graft tissue itself. UBLs may represent the sub-class of immune lymphocytes which induce necrosis of the grafted cells. Mitotic lymphocytes and cells resembling lymphocytes transformed by phytohaemagglutinin were also found in efferent homograft lymph.     

Human and Murine High Endothelial Venule Cells Phagocytose Apoptotic Leukocytes

Apoptotic cell death occurs during normal lymphocyte development and differentiation as well as following lymphocyte exposure to endogenous corticosteroids released during stress, malnutrition, and trauma. Recognition and engulfment of these apoptotic cells is important for the clearance of dying cells before they release potent inflammatory mediators into the vasculature or tissues. Phagocytosis of apoptotic cells is accomplished in part by macrophages. We report for the first time that apoptotic lymphocytes are also phagocytosed by high endothelial venule (HEV) cells. The murine HEV cell line mHEVa rapidly phagocytosed apoptotic lymphoid and myeloid cells with the greatest rate of phagocytosis occurring at 0–6 h. To confirm HEV cell interaction with apoptotic cells, we demonstrated that apoptotic human tonsil lymphocytes were phagocytosed by human tonsil HEV cells in primary cultures. Furthermore, we examined HEV cell phagocytosisin vivo.Mice were treated with a natural corticosterone (4-pregnene-11β,21-diol-3,20-dione) at levels detected during stress or malnutrition (93–180 μg serum cortisol/dl). At 4–12 h posttreatment, apoptotic lymphocytes were present inside vacuoles of HEV cells in axillary lymph node tissue sections, as determined by transmission electron microscopy. These data suggest that, in addition to macrophages, lymph node HEV cells also play a role in the removal of apoptotic lymphocytes. Moreover, since HEV cells are specialized endothelial cells that regulate lymphocyte migration into peripheral lymphoid tissues, they may provide an important checkpoint for clearance of apoptotic lymphocytes within the vasculature, as well as limiting entrance of nonfunctional lymphocytes into the lymph node.     

Effect of endotoxin on diaphragm lymph contamination in unanesthetized sheep

The preparation for collecting lung lymph from sheep caudal mediastinal lymph node (CMN) efferent vessels is widely used to study the effects of endotoxin on lung microvascular permeability. However, there are nonpulmonary lymph vessels that drain into the CMN along with the afferent lymph vessels from the lung. Thus CMN lymph is a mixture of lymph from the lung and diaphragm lymph vessels as well as from other nonpulmonary lymph vessels. We studied the effect of 0.5-1.0 microgram/kg Escherichia coli endotoxin on the flow rates in diaphragm and CMN efferent lymph vessels (Qdi and QCMN, respectively) in unanesthetized sheep. For the time period between 2 and 5.5 h after endotoxin QCMN was increased from its base line of 7.2 +/- 4.4 (SD) to 17.3 +/- 10.6 ml/h and the lymph-to-plasma protein concentration ratio (L/PCMN) had increased from 0.68 +/- 0.11 to 0.81 +/- 0.06. During the same time period, Qdi was 4.5 +/- 3.1 ml/h compared with 1.0 +/- 0.8 ml/h at base line and the diaphragm lymph-to-plasma protein concentration ratio (L/Pdi) was 0.92 +/- 0.07 (base line = 0.74 +/- 0.15). The increases in flow rate and protein concentration were significant for each type of vessel (P less than 0.05). We conclude that the period of increased QCMN and L/PCMN after endotoxin is associated with an increase in Qdi and L/Pdi. Thus, it is difficult to determine how much of the CMN lymph response comes from the lungs and how much comes from diaphragm lymph vessels.     

Dissemination of bovine leukemia virus-infected cells from a newly infected sheep lymph node

To investigate the early establishment of bovine leukemia virus (BLV) infection, we injected BLV-infected or mock-infected allogeneic cells into the shoulder of sheep in which an efferent lymphatic duct of the draining prescapular lymph node had been cannulated. Rare mononuclear cells acting as centers of BLV infection in culture were present within 4 to 6 days in efferent lymph and within 6 to 10 days in blood. Soon after BLV injection, immunoglobulin M+ (IgM+) and CD8+ cells increased in efferent lymph and oscillated reciprocally in frequency. CD8+ blasts increased on days 4 to 6, when infectious centers increased 100-fold in lymph. On days 6 and 7, both lymph and blood were enriched with CD8+ cells that were labeled late on day 5 with an intravenous pulse of 5-bromo-2'-deoxyuridine (BrdU). Lymph, but not blood, was enriched with BrdU+ B cells on day 7. Capsid-specific antibodies became detectable in efferent lymph on days 6 to 8 and surface glycoprotein-specific antibodies on day 9, preceding their detection in serum by 9 to 14 days. Systemic dissemination of BLV-infected cells was thus accompanied by an increase in proliferating CD8+ cells and the onset of BLV-specific antibodies in lymph. Infectious centers reached maximum frequencies of 0.2% in lymph by days 11 to 13, and then their frequencies increased by 5- to 40-fold in blood cells, suggesting that many infected blood cells do not recirculate back into lymph. Beginning on days 10 to 13, a subpopulation of B cells having high levels of surface IgM increased sharply in peripheral blood. Such cells were not present in lymph. After a day 16 pulse of BrdU, recently proliferated cells that stained intensely for surface IgM appeared in blood within 15 h. Predominantly B lymphocytes contained the viral capsid protein when lymph and blood cells were cultured briefly to allow BLV expression. However, both early in lymph and later in blood, BrdU+ B cells greatly exceeded productively infected cells, indicating that new BLV infections stimulate proliferation of two different populations of B cells.     

Intrathoracic sources of caudal mediastinal lymph node efferent lymph in sheep

We investigated the intrathoracic contributions to the caudal mediastinal lymph node (CMN) efferent lymph in 12 anesthetized sheep after removing possible systemic contributions from below the diaphragm. We interrupted various pathways that may send lymph to the CMN (chest wall, esophagus, lung). Because the experiment is destructive, we did the resections in various combinations and waited 1 h between steps. Base-line CMN efferent lymph flow averaged 0.90 +/- 0.52 g/15 min (mean +/- SD). Cutting the pulmonary ligaments bilaterally caused lymph flow to decrease by an average of 58%. In five sheep, cauterizing around the lung hila reduced lymph flow by 16% of base line, cauterizing along the esophagus reduced lymph flow by 18% of base line, and cauterizing along the chest wall increased lymph flow by 6% of base line. After complete isolation of the node, except for the bronchoesophageal artery, dorsal mediastinal vein, and CMN efferent duct, 14% of base-line flow remained. The lymph-to-plasma total protein concentration ratios did not change significantly with any procedure. Under the conditions of our experiments, approximately 74% of base-line intrathoracic CMN efferent flow comes from the lung.     

Ontogeny of the antigen-reactive lymph follicle-forming capacity of the popliteal lymph node in neonatal mice

The ontogenetic development of the reactive lymph follicle-forming capacity of the popliteal lymph node was investigated immunohistochemically in young mice which had received a single injection of hemocyanin (KLH) in a rear footpad at a predetermined age (between 1 and 21 days). The mice were sacrificed at various intervals after injection. In non-stimulated young mice, primary lymph follicles first appeared in the popliteal node at 11 days of age. When KLH was given to 7-day-old or older mice, each draining popliteal node showed a marked increase in B lymphocytes in the extrafollicular zone 3 days after injection and produced a number of "new" lymph follicles outside the pre-existing follicles over the next few days. In mice injected at 2-4 days of age, these nodes showed an increase in B lymphocytes in the outer cortex and had produced several lymph follicles by 8 days of age. The number of lymph follicles produced by each node tended to increase in line with age at injection. These results indicate that neonatal popliteal nodes become able to produce lymph follicles in response to exogenous antigens some time before ontogenetically developing follicles appear. The formation of new lymph follicles observed in draining popliteal nodes after KLH injection at an early postnatal age is discussed in relation to the ontogenetic development of stromal cells (precursors of follicular dendritic cells) that are capable of interacting with B lymphocytes and the extent of B lymphocyte influx into the node induced by KLH stimulation.     

Focal topographic changes in inflammatory microcirculation associated with lymphocyte slowing and transmigration

Microcirculation is the primary mechanism for delivering lymphocytes to inflammatory tissues. Blood flow within microvessels ensures a supply of lymphocytes at the blood-endothelial interface. Whether the structure of the inflammatory microcirculation facilitates lymphocyte transmigration is less clear. To illuminate the microcirculatory changes associated with lymphocyte transmigration, we used intravital videomicroscopy to examine the dermal microcirculation after application of the epicutaneous antigen oxazolone. Intravascular injection of fluorescein-labeled dextran demonstrated focal topographic changes in the microcirculation. These focal changes had the appearance of loops or hairpin turns in the oxazolone-stimulated skin. Changes were maximal at 96 h and coincided with peak lymphocyte recruitment. To determine whether these changes were associated with lymphocyte transmigration, lymphocytes obtained from efferent lymph of draining lymph nodes at 96 h were fluorescently labeled and reinjected into inflammatory microcirculation. Epifuorescence intravital video microscopy demonstrated focal areas were associated with lymphocyte slowing and occasional transmigration. In contrast, focal loops and lymphocyte slowing were rarely observed in the contralateral control microcirculation. Results suggest that structural adaptations in inflammatory microcirculation represented by focal topographic changes may contribute to regulation of tissue entry by recirculating lymphocytes.     

Lymphocyte and granulocyte migration across the endothelial layer of bovine pulmonary artery intimal explants towards lymphocyte conditioned medium

An intravenous infusion of endotoxin into sheep results in accumulation of equal numbers of lymphocytes and granulocytes in the pulmonary microcirculation. The role of the sequestered lymphocytes in acute lung injury is not known. The present study examines whether lymphocyte migration through pulmonary endothelium contributes to endothelial damage and also examines the effect of lymphokines on granulocyte migration. Bovine pulmonary artery intimal explants were mounted in Boyden chambers and conditioned media, prepared from bovine peripheral blood lymphocytes, was used as the chemoattractant. The rate of 51Cr labelled bovine granulocyte lymphocyte migration into intimal explants was determined over a 3 hr incubation period. Permeability changes were assessed by adding trace amounts of 14C-sucrose and 3H-water to the upper well and following their rate of equilibration with the lower well. 6-Keto-PGF1 alpha was measured in the upper well. Lymphocyte conditioned media was found to be chemotactic for both lymphocytes and granulocytes (lymphocyte migration at 60 min: lymphocyte conditioned media = 18.5 +/- 2.3%, mean +/- s.e. RPMI control = 12.5 +/- 1.5; granulocyte migration at 120 min: conditioned media = 36.1 +/- 5.7, RPMI control = 18.2 +/- 3.0). Ultrastructural examination revealed leukocyte migration followed an orderly sequence during which the leukocytes maintained close contact with the adjacent endothelial cells. No structural evidence of endothelial cell damage was seen at any time examined. Granulocyte migration was associated with an increased rate of 14C-sucrose equilibration after 2 hr of incubation (lower well counts/upper well counts at 2 hr, RPMI control = 0.18 +/- 0.02; lymphocyte conditioned medium = 0.30 +/- 0.04) indicating alteration in the endothelial barrier function. Leukocyte migration, particularly lymphocyte migration, was accompanied by a marked increase in prostacyclin accumulation (3 hr: no leukocytes, 188 +/- 17 ng/ml; lymphocytes, 560 +/- 104). These in vitro findings suggest that lymphocytes and lymphokines may be involved in acute lung injury and also that permeability changes associated with granulocyte migration may depend on the chemoattractant.     

Epinephrine causes a reduction in lymph node cell output in sheep

The lymphatic system has a critical role in the return of fluids, proteins, and cells to the circulatory system. However, the effects of stress, including exercise, on this system have not been adequately studied. We investigated the effect of a physiological dose (1 mg) of epinephrine (Epi) on lymph flow, cell concentration, and lymphocyte subsets in efferent subcutaneous lymph in sheep. Blood leukocyte numbers, differential, lymphocyte subsets, and blood and lymph pools of lymphocytes were determined simultaneously. A significant acute increase in lymph flow was followed by a post-injection decrease in flow and cellular output. No changes in lymphocyte subsets or pools of lymphocytes were seen in either blood or lymph. The timing of elevated plasma and lymph concentrations of Epi and norepinephrine (NE) corresponded with the increased lymph flow. In conclusion, Epi injection caused no change in lymphocyte subset distribution, leukocyte concentration, or pools of lymphocytes. A decrease in lymph flow and cellularity was documented post-injection, indicating that lymphatic tissue has no role in the leukocytosis seen after Epi injection. Lymphocyte retention by lymph nodes, however, may contribute to post-injection lymphopenia.     

FOXP3+ Lymphocyte Density in Pancreatic Cancer Correlates with Lymph Node Metastasis

Objective

To determine if the density of FOXP3⁺ lymphocytes in primary tumors and lymph nodes in pancreatic cancer correlates with the presence of lymph node metastases.

Methods

FOXP3⁺ lymphocyte density in primary pancreatic cancer tissue and draining lymph nodes was measured using immunohistochemistry. We analyzed the clinical and pathological aspects associated with the accumulation of FOXP3⁺ lymphocytes in pancreatic cancer. We also analyzed the correlation of density of FOXP3⁺ lymphocytes in lymph nodes with the nodal status and distance from the primary tumor.

Results

FOXP3⁺ lymphocyte density in pancreatic cancer was significantly higher than in paratumoral pancreatic tissue. The density of FOXP3⁺ lymphocytes in local tumor tissue correlated significantly with the histological grade and overall lymph node status. Furthermore, FOXP3⁺ lymphocyte density was significantly higher in positive lymph nodes than in negative ones, while it had no correlation with the distance of the lymph node from the primary tumor.

Conclusion

FOXP3⁺ lymphocyte density in primary tumor tissue in patients with pancreatic cancer correlates with lymph node metastasis. Lymph nodes containing metastases having higher FOXP3⁺ lymphocyte densities than do negative lymph nodes.