Prognostic value of the expression of chemokines and their receptors in regional lymph nodes of melanoma patients

Chemokines and their receptors have been reported to drive immune cells into tumours or to be directly involved in the promotion or inhibition of the development of tumours. However, their expression in regional lymph node (LN) tissues in melanoma patients remains unknown. The present study investigated the relationship between the expression of mRNA of chemokines and their receptors and clinicopathology of the regional LN tissues of skin cutaneous melanoma (SKCM) patients available in The Cancer Genome Atlas. The relationship between chemokines and their receptors and the composition of immune cells within the tumour was analysed. In SKCM regional LN tissues, the high expression of 32 types of chemokines and receptors, namely CCL2, 4-5, 7-8, 13, 22-25, CCR1-9, CXCL9-13, 16, CXCR3, 5, 6, XCL1-2 and XCR1 in LN was associated with favourable patient prognosis. Conversely, high expression of CXCL17 was an indicator of poor prognosis. The expression of mRNA for CXCL9-11, 13, CXCR3, 6, CCL2, 4, 5, 7, 8, 25, CCR1, 2, 5, and XCL1, 2 in regional LN tissues was positively correlated with the fraction of CD8-positive T cells and M1 macrophages, and was negatively correlated with M0 macrophages. CCR4, 6-9, CCL13, 22, 23 and XCR1 were positively correlated with the fraction of memory B cells and naive T cells, and negatively correlated with M0 macrophages and resting mast cells, suggesting that chemokines and their receptors may affect the prognosis of patients by guiding immune cells into the tumour microenvironment to eliminate tumour cells.     

Depletion of macrophages and disappearance of postcapillary high endothelial venules in lymph nodes deprived of afferent lymphatic vessels

Summary The afferent lymphatic vessels of rat popliteal lymph nodes were interrupted, and the histological alterations in the lymph nodes occurring 1 to 14 weeks after operation were studied. One week after operation the number of macrophages was considerably reduced and continued to decrease during the subsequent time periods studied. A 6 weeks most macrophages had disappeared. Simultaneously the immunological activity diminished and had completely disappeared 8 weeks after operation. Three weeks after operation the endothelial cells of the postcapillary high endothelial venules had flattened, and the number of immigrating lymphocytes was greatly reduced. Subsequently the lymph nodes became depleted of both macrophages and lymphocytes, leaving only the reticuloendothelial framework.     

Extra-organic lymphatic vessels and regional lymph nodes of the kidneys in dogs

An anatomical investigation of extraorganic lymphatic vessels and regional renal lymph nodes has been performed in 70 dogs. The regional lymph nodes in the right kidney are stated to be quantitatively constant, as well as cranial and caudal lateroaortal lymph nodes in the left kidney in regard to the middle left lateroaortal nodes, that get lymph from the left kidney parenchyma. One middle left lateroaortal lymph node is found in 47 animals examined, two lymph nodes--in 17 animals. In 6 cases a lymphatic vessel, that gets lymph from the renal parenchyma and independently runs into the cistern of the thoracic duct is found for the first time. The variant revealed is an exception from the rule known in lymphology: lymph in its way from periphery to the central collector runs, at least, through one lymph node.     

IL-3 induces expression of lymphatic markers Prox-1 and podoplanin in human endothelial cells

Factors determining lymphatic differentiation in the adult organism are not yet well characterized. We have made the observation that mixed primary cultures of dermal blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) grown under standard conditions change expression of markers during subculture: After passage 6, they uniformly express LEC-specific markers Prox-1 and podoplanin. Using sorted cells, we show that LEC but not BEC constitutively express IL-3, which regulates Prox-1 and podoplanin expression in LEC. The addition of IL-3 to the medium of BEC cultures induces Prox-1 and podoplanin. Blocking IL-3 activity in LEC cultures results in a loss of Prox-1 and podoplanin expression. In conclusion, endogenous IL-3 is required to maintain the LEC phenotype in culture, and the addition of IL-3 to BEC appears to induce transdifferentiation of BEC into LEC.     

Anatomical variants of the lymphatic vessels connecting the inguinal lymph nodes]

The study of anatomical variants of lymphatic vessels connecting inguinal lymph nodes was carried out on 56 corpses of adult persons of both sex whose deaths were not connected with lesions in the lymphatic system of the pelvis and lower extremities. The inguinal lymph nodes and their afferent and efferent lymphatic vessels were detected by the method of intradermal injection and by the method of direct injection into the lymphatic vessels. It was stated that groups of the inguinal lymph nodes, as well as the nodes in every group determined, can serve as nodes of different stages for afferent lymphatic vessels running from different parts of the body and organs.     

Gene expression profile of lymphatic endothelial cells

The lymphatic system was first described at around the same time as the blood circulation centuries ago, but the biological function elucidation of LECs (lymphatic endothelial cells) is far less than that of BVECs (blood vascular endothelial cells). Since the discovery of molecular markers for LECs and exploration of lymphatic role in tumour metastasis, more attention has been given to basic lymphatic research. Approx. 150 known genes were found to be expressed at the mRNA and protein levels by LECs. These molecules play an important role in lymphangiogenesis, signalling, tumour metastasis, immune function and fluid transport. This review provides a brief outline of gene expression profile of LECs and the molecular biological function, which will give the reader a better understanding about the mechanics of lymphatic function and some pathologies related to the lymphatic system such as lymphoedema, and facilitate advanced scientific research into lymphatic biology.     

Interaction of lymphocytes and high endothelial venules in irradiated lymph nodes

After total-body exposure to various doses of ionizing radiation, the ability of lymphocytes to interact specifically with high endothelial venules of rat cervical and mesenteric lymph nodes was analyzed in frozen sections. Following a radiation dose of 1.5 Gy, high endothelial venules remained intact and the binding of unirradiated lymphocytes to the venules was enhanced relative to unirradiated controls. At radiation doses above 5.0 Gy, damage to high endothelial venules was observed histologically as well as assessed functionally. There was a significant decrease in specific lymphocyte-venule binding and a significant increase in nonspecific binding. These findings suggest that radiation-induced damage to high endothelial venules might play a role in radiation-induced immunosuppression by interfering with the normal passage of lymphocytes from the blood into lymph nodes via a specific interaction between lymphocytes and high endothelial venules.     

Dynamic imaging of lymphatic vessels and lymph nodes using a bimodal nanoparticulate contrast agent

BACKGROUND: Evaluation of lymphedema and lymph node metastasis in humans has relied primarily on invasive or radioactive modalities. While noninvasive technologies such as magnetic resonance imaging (MRI) offer the potential for true three-dimensional imaging of lymphatic structures, invasive modalities, such as optical fluorescence microscopy, provide higher resolution and clearer delineation of both lymph nodes and lymphatic vessels. Thus, contrast agents that image lymphatic vessels and lymph nodes by both fluorescence and MRI may further enhance our understanding of the structure and function of the lymphatic system. Recent applications of bimodal (fluorescence and MR) contrast agents in mice have not achieved clear visualization of lymphatic vessels and nodes. Here the authors describe the development of a nanoparticulate contrast agent that is taken up by lymphatic vessels to draining lymph nodes and detected by both modalities. METHODS: A unique nanoparticulate contrast agent composed of a polyamidoamine dendrimer core conjugated to paramagnetic contrast agents and fluorescent probes was synthesized. Anesthetized mice were injected with the nanoparticulates in the hind footpads and imaged by MR and fluorescence microscopy. High resolution MR and fluorescence images were obtained and compared to traditional techniques for lymphatic visualization using Evans blue dye. RESULTS: Lymph nodes and lymphatic vessels were clearly observed by both MRI and fluorescence microscopy using the bimodal nanoparticulate contrast agent. Characteristic tail-lymphatics were also visualized by both modalities. Contrast imaging yielded a higher resolution than the traditional method employing Evans blue dye. MR data correlated with fluorescence and Evans blue dye imaging. CONCLUSION: A bimodal nanoparticulate contrast agent facilitates the visualization of lymphatic vessels and lymph nodes by both fluorescence microscopy and MRI with strong correlation between the two modalities. This agent may translate to applications such as the assessment of malignancy and lymphedema in humans and the evaluation of lymphatic vessel function and morphology in animal models.     

Patterns of lymphatic drainage to individual thoracic and cervical lymph nodes in the rat

Colloidal carbon was used as tracer material to determine the lymphatic drainage to the cervical and thoracic lymph nodes from various regions of the respiratory tract in the F344 rat. While the lung region may be drained mainly to the posterior mediastinal lymph nodes, the tracheal wall drains primarily to the internal jugular and posterior cervical nodes.     

Oncostatin M enhances CCL21 expression by microvascular endothelial cells and increases the efficiency of dendritic cell trafficking to lymph nodes

CCL21, a lymphatic endothelial cell (LEC)-derived chemokine, and its receptor CCR7 regulate dendritic cell (DC) trafficking to lymph nodes (LN), but it is unclear how CCL21 expression is regulated. Oncostatin M (OSM) is an IL-6-like cytokine synthesized by activated DC and other leukocytes. In vitro, OSM (but not TNF-alpha) stimulated CCL21 mRNA and protein expression by human dermal microvascular EC (DMEC) in an ERK1/2-dependent fashion. Conditioned medium from OSM-treated DMEC stimulated CCL21-dependent chemotaxis of mouse bone marrow-derived DC (BMDC). Cultured BMDC expressed OSM, which was increased with the addition of LPS. Topical application of the contact-sensitizing hapten, trinitrochlorobenzene, resulted in enhanced OSM expression in the skin, whereas cutaneous injection of TNF-alpha did not. Injection of OSM into the footpad increased CCL21 mRNA expression in the draining LN by approximately 10-fold and in mouse skin by approximately 4-fold without increasing CCR7 mRNA. In vitro, OSM increased the permeability of DMEC and lung microvascular EC monolayers to FITC-dextran beads, and, in vivo, it enhanced accumulation of Evans blue dye in draining LN by approximately 3-fold (p = 0.0291). Of note, OSM increased trafficking of BMDC injected in footpads to draining LN by 2-fold (p = 0.016). In summary, OSM up-regulates CCL21 expression in skin and draining regional LN. We propose that OSM is a regulator of CCL21 expression and endothelial permeability in skin, contributing to efficient migration of DC to regional LN.     

Afferent and efferent connections of the area postrema demonstrated by the horseradish peroxidase method

The previous autoradiographic study of the efferent connections of the arca postrema have been completed by using horseradish peroxidase (H.R.P.) as a marking agent. These two labelling techniques have made it possible to determine two categories of structures connected with A.P.: those with afferent connections (Nucleus ambiguus), or efferent connections (mesencephalic nucleus of V, Locus coeruleus, griseum centrale and inferior and superior colliculi) and those having both types of connections (Nucleus tractus solitarii (N.F.S.), Dorsal vagal nucleus, nucleus intercalatus, hypoglossal nucleus). The afferent and efferent pathways of the A.P. identified with H.R.P. coincide with the catecholaminergic pathways demonstrated in the rat by Lindvall and Bj?rklund (1974).     

Topography of lymphatic collectors and regional lymph nodes of the canine heart and their morphological characteristics

In 40 dogs lymphatic vessels and regional lymph nodes of the heart have been prepared. Morphology of the regional lymph nodes have been studied by means of various histological techniques. Lymph outflow from the canine ventricles is realized by three (less often), or by two (more often) collectors. In very rare cases one collector is formed. From the right atrium lymph flows out in two collectors (cranial and left). Lymphatic vessels of the left atrium get into the left collector of the ventricles, or into the tracheobronchial lymph nodes. Into the same nodes gets the lymphatic vessel, forming at the border of the left and right atrii. Cranial, medial, caudal mediastinal nodes (lymphatic mediastinal system) and right, middle and left tracheobronchial lymph nodes (tracheobronchial system) are regional lymph nodes of the canine heart. In the lymph nodes of the tracheobronchial system of puppies older than one month presence of exogenic pigment and signs of fibrous degeneration of parenchyma are noted.     

High endothelial venules of the lymph nodes express Fas ligand.

Fas (CD95, APO-1) is widely expressed on lymphatic cells, and by interacting with its natural ligand (Fas-L), Fas induces apoptosis through a complex caspase cascade. In this study we sought to survey Fas-L expression in vascular and sinusoidal structures of human reactive lymph nodes. Immunohistochemical Fas-L expression was present in all paracortical high endothelial venules (HEVs), in cells lining the marginal sinus wall, and in a few lymphocytes, but only occasionally in non-HEV vascular endothelium. In the paracortical zone over 60% of all vessels and all paracortical HEVs showed Fas-L expression, whereas in the medullary zone less than 10% of the blood vessels were stained with Fas-L. Normal vessels outside lymph nodes mostly showed no Fas-L expression. We show that in human reactive lymph nodes Fas-L expression is predominantly present in HEVs. Because the circulating lymphocytes gain entry to nodal parenchyma by transendothelial migration through HEVs, the suggested physiological importance of Fas-L expression in these vessels lies in the regulation of lymphocyte access to lymph node parenchyma by possibly inducing Fas/Fas-L mediated apoptosis of activated Fas-expressing lymphoid cells. The Fas-L expressing cells in the marginal sinus might have a similar function for cells accessing the node in afferent lymph.     

Isolation and characterization of high endothelial cell lines derived from mouse lymph nodes

Summary Two long-term cultured cell lines were established from BALB/c mouse axillary and cervical lymph nodes that exhibited a combination of functional, morphological, and phenotypic characteristics consistent only with high endothelial venule cells. Spleen lymphocytes selectively bound and migrated across the cell lines. On Matrigel, these cell lines formed tubules with lumens, a characteristic unique to endothelial cells. Morphologically the cells were 20–30 μm in diameter and exhibited contact inhibition. The cells were not myeloid in origin because they lacked sodium fluoride-inhibitable nonspecific esterase activity, myeloperoxidase activity, and F4/80 antigen. The cell line phenotypes were compared to high endothelial venule (HEV) cells in tissue sections. HEV cells in lymph node tissue sections expressed endoglin, PECAM-1, ICAM-1, VCAM-1, laminin, fibronectin, collagen IV, H2K^d, MECA 79, MECA 325, and vWF. The cell lines expressed endoglin, VCAM-1, fibronectin, and H2K^d. The cell line derived from cervical lymph nodes also expressed laminin and H2D^d. Neither cell line expressed collagen IV, IA^d, ICAM-1, ICAM-2, dendritic cell antigen, or PECAM-1. They also did not express MECA antigens or intracellular vWF, consistent with reports of many cultured endothelial cells. To further substantiate cell line identification, antiserum generated against the cell lines bound specifically to HEV cells in frozen lymph node tissue sections and to both of the lymph node-derived cell lines but not control cell lines. Thus, the lymph node derived-cell lines expressed molecules found on HEV cellsin vivo and most importantly retained the functions of tubule formation, lymphocyte adhesion, and promotion of lymphocyte migration.     

Intestinal and peri-tumoral lymphatic endothelial cells are resistant to radiation-induced apoptosis

Radiation therapy is a widely used cancer treatment, but it is unable to completely block cancer metastasis. The lymphatic vasculature serves as the primary route for metastatic spread, but little is known about how lymphatic endothelial cells respond to radiation. Here, we show that lymphatic endothelial cells in the small intestine and peri-tumor areas are highly resistant to radiation injury, while blood vessel endothelial cells in the small intestine are relatively sensitive. Our results suggest the need for alternative therapeutic modalities that can block lymphatic endothelial cell survival, and thus disrupt the integrity of lymphatic vessels in peri-tumor areas.     

Memory T cells are enriched in lymph nodes of selectin-ligand-deficient mice

Fucosyltransferase-IV and -VII double knockout (FtDKO) mice reveal profound impairment in T cell trafficking to lymph nodes (LNs) due to an inability to synthesize selectin ligands. We observed an increase in the proportion of memory/effector (CD44(high)) T cells in LNs of FtDKO mice. We infected FtDKO mice with lymphocytic choriomeningitis virus to generate and track Ag-specific CD44(high)CD8 T cells in secondary lymphoid organs. Although frequencies were similar, total Ag-specific effector CD44(high)CD8 T cells were significantly reduced in LNs, but not blood, of FtDKO mice at day 8. In contrast, frequencies of Ag-specific memory CD44(high)CD8 T cells were up to 8-fold higher in LNs of FtDKO mice at day 60. Because wild-type mice treated with anti-CD62L treatment also showed increased frequencies of CD44(high) T cells in LNs, we hypothesized that memory T cells were preferentially retained in, or preferentially migrated to, FtDKO LNs. We analyzed T cell entry and egress in LNs using adoptive transfer of bone fide naive or memory T cells. Memory T cells were not retained longer in LNs compared with naive T cells; however, T cell exit slowed significantly as T cell numbers declined. Memory T cells were profoundly impaired in entering LNs of FtDKO mice; however, memory T cells exhibited greater homeostatic proliferation in FtDKO mice. These results suggest that memory T cells are enriched in LNs with T cell deficits by several mechanisms, including longer T cell retention and increased homeostatic proliferation.