Endothelial Venules In Lymph Nodes Research Articles

Formyl peptide receptors are involved in CTX-induced impairment of lymphocyte functions

Crotoxin (CTX) is a neurotoxin that is isolated from the venom of Crotalus durissus terrificus, which displays immunomodulatory, anti-inflammatory, and anti-tumoral effects. Previous research has demonstrated that CTX promotes the adherence of leukocytes to the endothelial cells in blood microcirculation and the high endothelial venules of lymph nodes, which reduces the number of blood cells and lymphocytes. Studies have also shown that these effects are mediated by lipoxygenase-derived mediators. However, the exact lipoxygenase-derived eicosanoid involved in the CTX effect on lymphocytes is yet to be characterized. As CTX stimulates lipoxin-derived mediators from macrophages and lymphocyte effector functions could be modulated by activating formyl peptide receptors, we aimed to investigate whether these receptors were involved in CTX-induced redistribution and functions of lymphocytes in rats. We used male Wistar rats treated with CTX to demonstrate that Boc2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe), an antagonist of formyl peptide receptors, prevented CTX-induced decrease in the number of circulating lymphocytes and increased the expression of the lymphocyte adhesion molecule LFA1. CTX reduced the T and B lymphocyte functions, such as lymphocyte proliferation in response to the mitogen Concanavalin A and antibody production in response to BSA immunization, respectively, which was prevented by the administration of Boc2. Importantly, mesenteric lymph node lymphocytes from CTX-treated rats showed an increased release of 15-epi-LXA4. These results indicate that formyl peptide receptors mediate CTX-induced redistribution of lymphocytes and that 15-epi-LXA4 is a key mediator of the immunosuppressive effects of CTX.

A Straightforward Method for 3D Visualization of B Cell Clusters and High Endothelial Venules in Lymph Nodes Highlights Differential Roles of TNFRI and -II

Whole mount tissue immunolabeling and imaging of complete organs has tremendous benefits in characterizing organ morphology. Here, we present a straightforward method for immunostaining, clearing and imaging of whole murine peripheral lymph nodes (PLNs) for detailed analysis of their architecture and discuss all procedures in detail in a step-by-step approach. Given the importance of tumor necrosis factor receptor (TNFR) signaling in development of PLNs we used TNFRI-/- and TNFRII-/- mice models as proof-of-concept for this technique by visualizing and analyzing structural changes in PLN B cell clusters and high endothelial venules (HEVs). Samples were subjected to de- and rehydration with methanol, labeled with antibodies for B cells, T cells and high endothelial venules (HEVs) and optically cleared using benzyl alcohol-benzyl benzoate. Imaging was done using LaVision light sheet microscope and analysis with Imaris software. Using these techniques, we confirmed previous findings that TNFRI signaling is essential for formation of individual B cell clusters. In addition, Our data suggest that TNFRII signaling is also to some extent involved in this process as TNFRII-/- PLNs had a B cell cluster morphology reminiscent of TNFRI-/- PLNs. Moreover, visualization and objective quantification of the complete PLN high endothelial vasculature unveiled reduced volume, length and branching points of HEVs in TNFRI-/- PLNs, revealing an earlier unrecognized contribution of TNFRI signaling in HEV morphology. Together, these results underline the potential of whole mount tissue staining and advanced imaging techniques to unravel even subtle changes in lymphoid tissue architecture.

Periarteriolar stroma cells guide T cells from the red to the white pulp in the spleen.

While both the spleen and lymph nodes are called secondary lymphoid tissues, how lymphocytes enter these tissues are quite different from each other. This is because the architecture of the two types of organs and the mode of lymphocyte migration into these organs are quite distinct. In the spleen, T cells are passively released in the blood flow from the arterioles in the red pulp and marginal zone area. In contrast, T cells in the blood are actively captured on high endothelial venules in lymph nodes by the coordinated actions of CCR7 and several adhesion molecules. A recent finding indicates that T cells, released in the red pulp and marginal zone areas, actively find their way to the white zone by utilizing the migration track created by periarteriolar stromal cells. This finding adds one more piece to our understanding of lymphocyte migration for effective adaptive immune responses in the spleen.

Neutrophils Recirculate through Lymph Nodes to Survey Tissues for Pathogens.

The adaptive immune function of lymph nodes is dependent on constant recirculation of lymphocytes. In this article, we identify neutrophils present in the lymph node at steady state, exhibiting the same capacity for recirculation. In germ-free mice, neutrophils still recirculate through lymph nodes, and in mice cohoused with wild microbiome mice, the level of neutrophils in lymph nodes increases significantly. We found that at steady state, neutrophils enter the lymph node entirely via L-selectin and actively exit via efferent lymphatics via an S1P dependent mechanism. The small population of neutrophils in the lymph node can act as reconnaissance cells to recruit additional neutrophils in the event of bacterial dissemination to the lymph node. Without these reconnaissance cells, there is a delay in neutrophil recruitment to the lymph node and a reduction in swarm formation following Staphylococcus aureus infection. This ability to recruit additional neutrophils by lymph node neutrophils is initiated by LTB4. This study establishes the capacity of neutrophils to recirculate, much like lymphocytes via L-selectin and high endothelial venules in lymph nodes and demonstrates how the presence of neutrophils at steady state fortifies the lymph node in case of an infection disseminating through lymphatics.

Changes in high endothelial venules in lymph nodes after vascularized and nonvascularized lymph node transfer in a murine autograft model.

Vascularized lymph node transfer (LNT) is gaining popularity in the treatment of lymphedema. However, it is unclear whether the vascularization of transferred lymph nodes (LNs) contributes to functional improvement. High endothelial venules (HEVs) are specialized vessels that allow lymphocytes to enter LNs. In this study, we compared the numbers of HEVs and lymphocytes in LNs after vascularized and nonvascularized LNT. Fifty mice were divided into three groups (group 1, pedicled vascularized LNT; group 2, pedicled nonvascularized LNT; group 3, free nonvascularized LNT). Afferent lymphatic reconnection was confirmed by patent blue staining. The transferred LNs were harvested 4 weeks after surgery. HEVs, B-cells, and T-cells were subjected to immunohistochemical staining and quantified. Afferent lymphatic reconnection was observed in 13 of 20 transferred LNs in group 1, 11 of 15 in group 2, and 7 of 15 in group 3. The ratio of dilated/total HEVs in transferred LNs with afferent lymphatic reconnection was significantly higher in group 1 than in groups 2 and 3. No significant differences in numbers of B-cells and T-cells were found in the transferred LNs. We found that more functional HEVs were preserved in cases with successful afferent lymphatic reconnection after vascularized LNT than after nonvascularized LNT.

Targeted Delivery of Immunomodulators to Lymph Nodes.

SUMMARYActive-targeted delivery to lymph nodes represents a major advance toward more effective treatment of immune-mediated disease. The MECA79 antibody recognizes peripheral node address in molecules expressed by high endothelial venules of lymph nodes. By mimicking lymphocyte trafficking to the lymph nodes, we have engineered MECA79-coated microparticles containing an immunosuppressive medication, tacrolimus. Following intravenous administration, MECA79-bearing particles showed marked accumulation in the draining lymph nodes of transplanted animals. Using an allograft heart transplant model, we show that targeted lymph node delivery of microparticles containing tacrolimus can prolong heart allograft survival with negligible changes in tacrolimus serum level. Using MECA79 conjugation, we have demonstrated targeted delivery of tacrolimus to the lymph nodes following systemic administration, with the capacity for immune modulation in vivo.

Lysophosphatidic acid receptors LPA4 and LPA6 differentially promote lymphocyte transmigration across high endothelial venules in lymph nodes.

Naive lymphocytes continuously migrate from the blood into lymph nodes (LNs) via high endothelial venules (HEVs). To extravasate from the HEVs, lymphocytes undergo multiple adhesion steps, including tethering, rolling, firm adhesion and transmigration. We previously showed that autotaxin (ATX), an enzyme that generates lysophosphatidic acid (LPA), is highly expressed in HEVs, and that the ATX/LPA axis plays an important role in the lymphocyte transmigration across HEVs. However, the detailed mechanism underlying this axis's involvement in lymphocyte transmigration has remained ill-defined. Here, we show that two LPA receptors, LPA4 and LPA6, are selectively expressed on HEV endothelial cells (ECs) and that LPA4 plays a major role in the lymphocyte transmigration across HEVs in mice. In the absence of LPA4 expression, lymphocytes accumulated heavily within the HEV EC layer, compared to wild-type (WT) mice. This accumulation was also observed in the absence of LPA6 expression, but it was less pronounced. Adoptive transfer experiments using WT lymphocytes revealed that the LPA4 deficiency in ECs specifically compromised the lymphocyte transmigration process, whereas the effect of LPA6 deficiency was not significant. These results indicate that the signals evoked in HEV ECs via the LPA4 and LPA6 differentially regulate lymphocyte extravasation from HEVs in the peripheral LNs.

The Autotaxin–LPA Axis Emerges as a Novel Regulator of Lymphocyte Homing and Inflammation

Lysophosphatidic acid (LPA) is a pleiotropic lipid molecule with potent effects on cell growth and motility. Major progress has been made in recent years in deciphering the mechanisms of LPA generation and how it acts on target cells. Most research has been conducted in other disciplines, but emerging data indicate that LPA has an important role to play in immunity. A key discovery was that autotaxin (ATX), an enzyme previously implicated in cancer cell motility, generates extracellular LPA from the precursor lysophosphatidylcholine. Steady-state ATX is expressed by only a few tissues, including high endothelial venules in lymph nodes, but inflammatory signals can upregulate ATX expression in different tissues. In this article, we review current thinking about the ATX/LPA axis in lymphocyte homing, as well as in models of allergic airway inflammation and asthma. New insights into the role of LPA in regulating immune responses should be forthcoming in the near future.

In vivo high spatiotemporal resolution visualization of circulating T lymphocytes in high endothelial venules of lymph nodes

Lymph nodes (LN) are major checkpoints for circulating T lymphocytes to recognize foreign antigens collected from peripheral tissue. High endothelial venule (HEV) in LN facilitates the effective transmigration of circulating T lymphocytes from the blood into LN. There have been many efforts to visualize T lymphocytes trafficking across HEV to understand the underlying mechanism. However, due to insufficient spatiotemporal resolution and the lack of an in vivo labeling method, clear visualization of dynamic behaviors of rapidly flowing T lymphocytes in HEV and their transmigration have been difficult. In this work, we adapted a custom-designed video-rate laser scanning confocal microscopy system to track individual flowing T lymphocytes in the HEV in real time in vivo. We demonstrate that the HEVs in LN can be clearly identified in vivo with its distinctive cuboidal morphology of endothelial cells fluorescently labeled by intravenously injected anti-CD31 antibody conjugated with Alexa fluorophore. By visualizing the adaptively transferred T lymphocytes, we successfully analyzed dynamic flowing behaviors of T lymphocytes and their transendothelial migration while interacting with the endothelial cells in HEV.

Dilated High Endothelial Venules in Lymph Nodes Simulating Metastatic Thyroid Carcinoma

When examining microscopically lymph nodes removed as part of the surgical therapy for thyroid carcinoma, one should be aware of the fact that high endothelial venules, when dilated and near the capsule, can lead to a wrong diagnosis of metastasis. We present a case in which such a situation arose.

T-cell-rich angiomatoid polypoid pseudolymphoma of the skin: a clinicopathologic study of 17 cases and a proposed nomenclature

We describe a series of previously unreported, distinctive, polypoid solitary T-cell-rich cutaneous pseudolymphomas. The clinicopathologic features were examined in 17 cases. Patient ages ranged from 16 to 71 years (mean = 38.5) with a female predominance (female : male = 14 : 3). All lesions, clinically diagnosed most often as pyogenic granuloma, presented as a solitary, polypoid, erythematous, papule ranging in size from 2.5 to 7.5 mm (mean = 5.8). Most occurred on the head and neck (7) and trunk (6) with other sites including the thigh (1), shoulder (1) and knee (1). A dense dermal infiltrate composed of mildly atypical lymphocytes with variable numbers of admixed plasma cells and histiocytes was prototypical. Commonly, there was an associated epidermal collarette (16/17), Grenz zone (11/17) or admixed eosinophils (8/17). Prominent vessels lined by plump endothelial cells, reminiscent of high endothelial venules of lymph nodes, were universal and some degree of telangiectasia was also common (12/17). CD3-positive T-cells consisted of an admixture of CD4-positive and CD8-positive forms (15/16). Multiple studies suggested polyclonality (seven cases). No recurrences after lesional excision were noted in the 17 patients with a follow-up range from 24 to 120 months (mean = 46.6). Although these lesions share histopathologic features of the so-called acral pseudolymphomatous angiokeratoma of children (APACHE), they occur in a completely different clinical setting, present in solitary and polypoid fashion and are T-cell rich. We propose the diagnostic label T-cell-rich angiomatoid polypoid pseudolymphoma for this distinctive but presumably reactive lesion.

Microanatomy of lymphocyte-endothelial interactions at the high endothelial venules of lymph nodes.

Lymphocyte trafficking into lymph nodes and Peyer's patches is mediated primarily by specifically differentiated venules, called high endothelial venules (HEVs), located in the tissue parenchyma. HEVs have a unique morphology and phenotype, which enables them to interact with circulating lymphocytes efficiently. That is, the HEV endothelial cells have a tall and plump appearance, and constitutively express multiple adhesion molecules and chemokines on their surface. These molecules can interact with cognate receptors on circulating lymphocytes, thereby mediating the stepwise and sequential lymphocyte adhesion and transendothelial migration (TEM) at the HEV endothelial luminal surface. This review summarizes the fine morphological aspects of the unique HEV endothelial cells, with special reference to the spatial distribution of the adhesion molecules and chemokines that regulate lymphocyte migration.

296 Macrophage migration in tolerance

Active-targeted delivery to lymph nodes represents a major advance toward more effective treatment of immune-mediated disease. The MECA79 antibody recognizes peripheral node addressin molecules expressed by high endothelial venules of lymph nodes. By mimicking lymphocyte trafficking to the lymph nodes, we have engineered MECA79-coated microparticles containing an immunosuppressive medication, tacrolimus. Following intravenous administration, MECA79-bearing particles showed marked accumulation in the draining lymph nodes of transplanted animals. Using an allograft heart transplant model, we show that targeted lymph node delivery of microparticles containing tacrolimus can prolong heart allograft survival with negligible changes in tacrolimus serum level. Using MECA79 conjugation, we have demonstrated targeted delivery of tacrolimus to the lymph nodes following systemic administration, with the capacity for immune modulation in vivo.

α5β1 Integrin Blockade Inhibits Lymphangiogenesis in Airway Inflammation

The integrin alpha5beta1 has been previously implicated in tumor angiogenesis, but its role in the remodeling of both blood vessels and lymphatics during inflammation is at an early stage of understanding. We examined this issue using a selective, small-molecule inhibitor of alpha5beta1 integrin, 2-aroylamino-3-{4-[(pyridin-2-ylaminomethyl)heterocyclyl]phenyl}propionic acid (JSM8757), in a model of sustained airway inflammation in mice with Mycoplasma pulmonis infection, which is known to be accompanied by robust blood vessel remodeling and lymphangiogenesis. The inhibitor significantly decreased the proliferation of lymphatic endothelial cells in culture and the number of lymphatic sprouts and new lymphatics in airways of mice infected for 2 weeks but did not reduce remodeling of blood vessels in the same airways. In inflamed airways, alpha5 integrin immunoreactivity was present on lymphatic sprouts, but not on collecting lymphatics or blood vessels, and was not found on any lymphatics of normal airways. Macrophages, potential targets of the inhibitor, did not have alpha5 integrin immunoreactivity in inflamed airways. In addition, macrophage recruitment, assessed in infected airways by quantitative reverse transcription-polymerase chain reaction measurements of expression of the marker protein ionized calcium-binding adapter molecule 1 (Iba1), was not reduced by JSM8757. We conclude that inhibition of the alpha5beta1 integrin reduces lymphangiogenesis in inflamed airways after M. pulmonis infection because expression of the integrin is selectively increased on lymphatic sprouts and plays an essential role in lymphatic growth.

N-acetylglucosamine-6-O-sulfotransferases 1 and 2 cooperatively control lymphocyte homing through L-selectin ligand biosynthesis in high endothelial venules

Lymphocyte homing is mediated by specific interactions between L-selectin on lymphocytes and sulfated carbohydrates restricted to high endothelial venules in lymph nodes. Here we generated mice deficient in both N-acetylglucosamine-6-O-sulfotransferase 1 (GlcNAc6ST-1) and GlcNAc6ST-2 and found that mutant mice had approximately 75% less homing of lymphocytes to the peripheral lymph nodes than did wild-type mice. Consequently, these mice had lower contact hypersensitivity responses than those of wild-type mice. Carbohydrate structural analysis showed that 6-sulfo sialyl Lewis X, a dominant ligand for L-selectin, was almost completely absent from the high endothelial venules of these mutant mice, whereas the amount of unsulfated sialyl Lewis X was much greater. These results demonstrate the essential function of GlcNAc6ST-1 and GlcNAc6ST-2 in L-selectin ligand biosynthesis in high endothelial venules and their importance in immune surveillance.

Therapeutic Targeting of Endothelial Ligands for L-selectin (PNAd) in a Sheep Model of Asthma

The homing of lymphocytes to peripheral lymph nodes is initiated by an adhesive interaction between L-selectin on lymphocytes and PNAd, a set of sialomucins that are constitutively displayed on high endothelial venules of lymph nodes. PNAd is defined by monoclonal antibody MECA-79 that recognizes a sulfated oligosaccharide carried by the sialomucins. This epitope overlaps with 6-sulfo sialyl Lewis x, a recognition determinant for L-selectin. Previous work has shown that administration of a L-selectin monoclonal antibody blocks both late-phase airway responses and airway hyperresponsiveness in a sheep model of asthma. We show here that airway-associated lymphoid collections from lungs of allergic sheep exhibited PNAd(+) venules as detected by immunostaining with MECA-79. The same vessels also expressed a GlcNAc-6-O-sulfotransferase known as HEC-GlcNAc6ST, which is known to contribute to the formation of the MECA-79 epitope in high endothelial venules of mouse lymph nodes. Intravenous administration of MECA-79 to allergic sheep significantly blunted both the late-phase airway response and airway hyperresponsiveness induced by airway allergen challenge. Furthermore, MECA-79 inhibited the accumulation of all classes of leukocytes in bronchoalveolar lavage fluid. These findings represent the first demonstration that targeting of PNAd has therapeutic efficacy in an inflammatory disease.

Detection of a Sulfotransferase (HEC-GlcNAc6ST) in High Endothelial Venules of Lymph Nodes and in High Endothelial Venule-Like Vessels within Ectopic Lymphoid Aggregates: Relationship to the MECA-79 Epitope

The interaction of L-selectin on lymphocytes with sulfated ligands on high endothelial venules (HEVs) of lymph nodes results in lymphocyte rolling and is essential for lymphocyte homing. The MECA-79 monoclonal antibody reports HEV-expressed ligands for L-selectin by recognizing a critical sulfation-dependent determinant on these ligands. HEC-GlcNAc6ST, a HEV-localized sulfotransferase, is essential for the elaboration of functional ligands within lymph nodes, as well as the generation of the MECA-79 epitope. Here, we use an antibody against murine HEC-GlcNAc6ST to study its expression in relationship to the MECA-79 epitope. In lymph nodes, the enzyme is expressed in the Golgi apparatus of high endothelial cells, in close correspondence with luminal staining by MECA-79. In lymph node HEVs of HEC-GlcNAc6ST-null mice, luminal staining by MECA-79 is almost abolished, whereas abluminal staining persists although reduced in intensity. HEV-like vessels in several examples of inflammation-associated lymphoid neogenesis, including nonobese diabetic mice, also exhibit concomitant expression of the sulfotransferase and luminal MECA-79 reactivity. The correlation extends to ectopic lymphoid aggregates within the pancreas of RIP-BLC mice, in which CXCL13 is expressed in islets. Analysis of the progeny of RIP-BLC by HEC-GlcNAc6ST-null mice establishes that the enzyme is responsible for the MECA-79 defined luminal ligands.

Cutting edge: the B cell chemokine CXC chemokine ligand 13/B lymphocyte chemoattractant is expressed in the high endothelial venules of lymph nodes and Peyer's patches and affects B cell trafficking across high endothelial venules.

While CCR7 ligands direct T cell trafficking into lymph nodes (LNs) and Peyer's patches (PPs), chemokines that regulate B cell trafficking across high endothelial venules (HEVs) remain to be fully elucidated. Here we report that CXC chemokine ligand (CXCL)13 (B lymphocyte chemoattractant) is detected immunohistologically in the majority of HEVs in LNs and PPs of nonimmunized mice. Systemically administered anti-CXCL13 Ab bound to the surface of approximately 50% of HEVs in LNs and PPs, but not to other types of blood vessels, indicating that CXCL13 is expressed in the HEV lumen. In CXCL13-null mice, B cells rarely adhered to PP HEVs, whereas T cells did efficiently. Superfusion of CXCL13-null PPs with CXCL13 restored the luminal presentation of CXCL13 and also B cell arrest in PP HEVs at least partially. Collectively, these results indicate that CXCL13 expressed in the HEV lumen plays a crucial role in B cell trafficking into secondary lymphoid tissues such as PPs.

Increased chemokine receptor CCR7/EBI1 expression enhances the infiltration of lymphoid organs by adult T-cell leukemia cells

Adult T-cell leukemia (ATL) is characterized by infiltration of various tissues by circulating ATL cells, a finding often associated with a poor prognosis. Leukocyte migration from the circulation into tissues depends on integrin-mediated adhesion to the endothelium, and integrins are tightly regulated by several factors, such as chemokines. In this study, we focused on the interaction between chemokines and chemokine receptors on ATL cells to understand factors involved in ATL cell infiltration of lymphoid organs. We compared freshly isolated ATL cells from patients with and without lymphoid organ involvement for the expression of the chemokine receptor CCR7/EBI1, the functional receptor for secondary lymphoid-tissue chemokine (SLC), which is expressed at high levels by high endothelial venules of lymph nodes and Peyer's patches. Reverse transcriptase-polymerase chain reaction and flow cytometric analysis, using anti-CCR7 monoclonal antibody (CCR7.6B3), revealed that ATL cells from patients with lymphoid organ involvement expressed significantly more CCR7/EBI1 than control CD4+CD45RO+ T cells and ATL cells from patients without lymphoid organ involvement. Consequently, significantly more ATL cells from patients with lymphoid organ involvement than control CD4+CD45RO+ T cells and ATL cells from patients without lymphoid organ involvement adhered to surfaces coated with ICAM-1 and SLC or EBI1-ligand chemokine (ELC), another ligand for CCR7/EBI1, under static and flow conditions and migrated toward SLC or ELC at a low concentration (30 ng/ml). These findings suggest that increased CCR7/EBI1 expression plays a role in lymphoid organ infiltration of ATL cells. (Blood. 2000; 30-38)

Increased chemokine receptor CCR7/EBI1 expression enhances the infiltration of lymphoid organs by adult T-cell leukemia cells

AbstractAdult T-cell leukemia (ATL) is characterized by infiltration of various tissues by circulating ATL cells, a finding often associated with a poor prognosis. Leukocyte migration from the circulation into tissues depends on integrin-mediated adhesion to the endothelium, and integrins are tightly regulated by several factors, such as chemokines. In this study, we focused on the interaction between chemokines and chemokine receptors on ATL cells to understand factors involved in ATL cell infiltration of lymphoid organs. We compared freshly isolated ATL cells from patients with and without lymphoid organ involvement for the expression of the chemokine receptor CCR7/EBI1, the functional receptor for secondary lymphoid-tissue chemokine (SLC), which is expressed at high levels by high endothelial venules of lymph nodes and Peyer's patches. Reverse transcriptase-polymerase chain reaction and flow cytometric analysis, using anti-CCR7 monoclonal antibody (CCR7.6B3), revealed that ATL cells from patients with lymphoid organ involvement expressed significantly more CCR7/EBI1 than control CD4+CD45RO+ T cells and ATL cells from patients without lymphoid organ involvement. Consequently, significantly more ATL cells from patients with lymphoid organ involvement than control CD4+CD45RO+ T cells and ATL cells from patients without lymphoid organ involvement adhered to surfaces coated with ICAM-1 and SLC or EBI1-ligand chemokine (ELC), another ligand for CCR7/EBI1, under static and flow conditions and migrated toward SLC or ELC at a low concentration (30 ng/ml). These findings suggest that increased CCR7/EBI1 expression plays a role in lymphoid organ infiltration of ATL cells. (Blood. 2000; 30-38)