Lymphocyte Transendothelial Migration Research Articles

TET2-mediated tumor cGAS triggers endothelial STING activation to regulate vasculature remodeling and anti-tumor immunity in liver cancer

Induction of tumor vascular normalization is a crucial measure to enhance immunotherapy efficacy. cGAS-STING pathway is vital for anti-tumor immunity, but its role in tumor vasculature is unclear. Herein, using preclinical liver cancer models in Cgas/Sting-deficient male mice, we report that the interdependence between tumor cGAS and host STING mediates vascular normalization and anti-tumor immune response. Mechanistically, TET2 mediated IL-2/STAT5A signaling epigenetically upregulates tumor cGAS expression and produces cGAMP. Subsequently, cGAMP is transported via LRRC8C channels to activate STING in endothelial cells, enhancing recruitment and transendothelial migration of lymphocytes. In vivo studies in male mice also reveal that administration of vitamin C, a promising anti-cancer agent, stimulates TET2 activity, induces tumor vascular normalization and enhances the efficacy of anti-PD-L1 therapy alone or in combination with IL-2. Our findings elucidate a crosstalk between tumor and vascular endothelial cells in the tumor immune microenvironment, providing strategies to enhance the efficacy of combinational immunotherapy for liver cancer.

Single-cell transcriptome profiling reveals immune and stromal cell heterogeneity in primary Sjögren’s syndrome

Primary Sjögren's syndrome (pSS) is a complex autoimmune disease characterized by lymphocytic infiltration and exocrine dysfunction, particularly affecting the salivary gland (SG). We employed single-cell RNA sequencing to investigate cellular heterogeneity in 11 patients with pSS and 5 non-SS controls. Notably, patients with pSS exhibited downregulated SOX9 in myoepithelial cells, potentially associated with impaired epithelial regeneration. An expanded ACKR1+ endothelial subpopulation in patients with pSS suggested a role in facilitating lymphocyte transendothelial migration. Our analysis of immune cells revealed expanded IGHD+ naive B cells in peripheral blood from patients with pSS. Pseudotime trajectory analysis outlined a bifurcated differentiation pathway for peripheral B cells, enriching three subtypes (VPREB3+ B, BANK1+ B, CD83+ B cells) within SGs in patients with pSS. Fibroblasts emerged as pivotal components in a stromal-immune interaction network, potentially driving extracellular matrix disruption, epithelial regeneration impairment, and inflammation. Our study illuminates immune and stromal cell heterogeneity in patients with pSS, offering insights into therapeutic strategies.

Studies in humans and mice reveal a critical role for CCR2 in trafficking of pDCs during infection with SARS-CoV-2

Abstract Although GWAS have implicated chemokine receptors in affecting disease severity, the role of chemokines in COVID-19 pathogenesis has not been defined. To bridge this gap, we surveyed expression of 14 chemokine receptors on blood leukocytes from 19 hospitalized COVID-19 patients vs. healthy controls. Among many differences between patients and controls, we found that CCR2, which we have shown mediates lymphocyte transendothelial migration, was down on patient MAIT cells and pDCs, whose numbers in blood are depressed in COVID-19. If a chemokine receptor mediates extravasation, lower levels of the receptor on blood cells could result from efficient, and thereby preferential migration of leukocytes expressing higher levels of the receptor into infected tissue. In support of this mechanism, we found enrichment of CCR2 highcells among MAIT cells migrating to the CCR2 ligand, CCL2, in vitro. To test if CCR2 mediates leukocyte extravasation in COVID-19 we infected transgenic K18-hACE2 Ccr2 reporter mice that were Ccr2 +/RFP(Ccr2 sufficient) or Ccr2 RFP/RFP(Ccr2 deficient) with SARS-CoV-2 intranasally and analyzed pDCs. Blood pDCs were RFP +and their numbers were decreased in Ccr2 RFP/RFPmice at baseline. Over seven days after infection with SARS-CoV-2 (LD 50), pDCs increased and then decreased in the blood, while increasingly progressively in lungs in both Ccr2 +/RFPand Ccr2 RFP/RFPmice. However, absolute numbers of lung pDCs and ratios of lung/blood pDCs were markedly diminished, as was survival, in Ccr2 RFP/RFPmice. We propose that increased mortality in Ccr2-deficient mice may be due in part to inadequate numbers of lung pDCs, leading to local deficiencies in type 1 and III interferons, which are critical for defense against SARS-CoV-2.

Trans-Endothelial Migration of Memory T Cells Is Impaired in Alemtuzumab-Treated Multiple Sclerosis Patients.

The breakdown of the blood-brain barrier (BBB) and the trans-endothelial migration of lymphocytes are central events in the development of multiple sclerosis (MS). Autoreactive T cells are major players in MS pathogenesis, which are rapidly depleted following alemtuzumab treatment. This modulation, in turn, inhibits CNS inflammation, but alemtuzumab's effect on T cell migration into the CNS has been less studied. Human brain endothelial cells were stimulated with pro-inflammatory cytokines to mimic an inflamed BBB in vitro. Peripheral blood mononuclear cells from healthy controls, untreated or alemtuzumab-treated patients with relapsing-remitting MS (RRMS) were added to the BBB model to assess their transmigratory capacity. Here, the migration of CD4+ effector memory T (TEM) and CD8+ central memory T (TCM) cells across the BBB was impaired in alemtuzumab-treated patients. Naïve T (Tnaïve) cells were unable to migrate across all groups. CD38 was lowly expressed on CD8+ TCM cells, particularly for RRMS patients, compared to CD8+ Tnaïve cells. CD62L expression was lower on CD4+ TEM cells than CD4+ Tnaïve cells and decreased further in alemtuzumab-treated patients. These data suggest that repopulated memory T cells are phenotypically different from naïve T cells, which may affect their transmigration across the BBB in vitro.

HIV-1 Tat and Heparan Sulfate Proteoglycans Orchestrate the Setup of in Cis and in Trans Cell-Surface Interactions Functional to Lymphocyte Trans-Endothelial Migration.

HIV-1 transactivating factor Tat is released by infected cells. Extracellular Tat homodimerizes and engages several receptors, including integrins, vascular endothelial growth factor receptor 2 (VEGFR2) and heparan sulfate proteoglycan (HSPG) syndecan-1 expressed on various cells. By means of experimental cell models recapitulating the processes of lymphocyte trans-endothelial migration, here, we demonstrate that upon association with syndecan-1 expressed on lymphocytes, Tat triggers simultaneously the in cis activation of lymphocytes themselves and the in trans activation of endothelial cells (ECs). This “two-way” activation eventually induces lymphocyte adhesion and spreading onto the substrate and vascular endothelial (VE)-cadherin reorganization at the EC junctions, with consequent endothelial permeabilization, leading to an increased extravasation of Tat-presenting lymphocytes. By means of a panel of biochemical activation assays and specific synthetic inhibitors, we demonstrate that during the above-mentioned processes, syndecan-1, integrins, FAK, src and ERK1/2 engagement and activation are needed in the lymphocytes, while VEGFR2, integrin, src and ERK1/2 are needed in the endothelium. In conclusion, the Tat/syndecan-1 complex plays a central role in orchestrating the setup of the various in cis and in trans multimeric complexes at the EC/lymphocyte interface. Thus, by means of computational molecular modelling, docking and dynamics, we also provide a characterization at an atomic level of the binding modes of the Tat/heparin interaction, with heparin herein used as a structural analogue of the heparan sulfate chains of syndecan-1.

CCL4 induces inflammatory signalling and barrier disruption in the neurovascular endothelium

BackgroundDuring neuroinflammation many chemokines alter the function of the blood-brain barrier (BBB) that regulates the entry of macromolecules and immune cells into the brain. As the milieu of the brain is altered, biochemical and structural changes contribute to the pathogenesis of neuroinflammation and may impact on neurogenesis. The chemokine CCL4, previously known as MIP-1β, is upregulated in a wide variety of central nervous system disorders, including multiple sclerosis, where it is thought to play a key role in the neuroinflammatory process. However, the effect of CCL4 on BBB endothelial cells (ECs) is unknown. Materials and methodsExpression and distribution of CCR5, phosphorylated p38, F-actin, zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-cadherin) were analysed in the human BBB EC line hCMEC/D3 by Western blot and/or immunofluorescence in the presence and absence of CCL4. Barrier modulation in response to CCL4 using hCMEC/D3 monolayers was assessed by measuring molecular flux of 70 ​kDa RITC-dextran and transendothelial lymphocyte migration. Permeability changes in response to CCL4 in vivo were measured by an occlusion technique in pial microvessels of Wistar rats and by fluorescein angiography in mouse retinae. ResultsCCR5, the receptor for CCL4, was expressed in hCMEC/D3 cells. CCL4 stimulation led to phosphorylation of p38 and the formation of actin stress fibres, both indicative of intracellular chemokine signalling. The distribution of junctional proteins was also altered in response to CCL4: junctional ZO-1 was reduced by circa 60% within 60 ​min. In addition, surface VE-cadherin was redistributed through internalisation. Consistent with these changes, CCL4 induced hyperpermeability in vitro and in vivo and increased transmigration of lymphocytes across monolayers of hCMEC/D3 cells. ConclusionThese results show that CCL4 can modify BBB function and may contribute to disease pathogenesis.

INDIVIDUAL FEATURES OF THE RESPONSE OF THE HUMAN IMMUNITY SYSTEM TO A SHORT-TERM STAY IN AIRCOOLED TO -25°C

The study of the influence of cold on the individual characteristics of the response of the human immune system is of great importance from the standpoint of ensuring the adaptive reactions of a person’s stay among extreme environmental factors. Purpose of the study. To establish the individual characteristics of the response of the human immune system to a short-term stay in an air environment cooled to -25°C. Materials and methods. A complex of immunological studies of the composition of the peripheral venous blood of the examined practically healthy volunteers (132 women and 40 men) living in the city of Arkhangelsk, aged from 21 to 50 years, was carried out. Blood was collected from the cubital vein on an empty stomach in the morning before and immediately after total cooling for 5 minutes at a temperature of -25°C. Results. It was found that 14.53% of the examined people are sensitive to general cooling and have an initial high level of background activity of the immune system. These individuals reacted by decreasing the level of lymphocytes in the blood by 1.5-4.5 times due to all functional forms of lymphocytes and by increasing the concentration of ATP in lymphocytes. A decrease in the lymphocyte count in response to general cooling is associated with an increase in the concentration of pro-inflammatory IL-6 and cortisol without changes in the content of IL-1β, IL-4 and IL-10. Conclusion. The effect of cold on the human body leads to a decrease in the number of lymphocytes in the venous blood in 14.53% of cases, which can be explained by a decrease in the blood flow velocity in the vascular bed and the transition of lymphocytes to the parietal pool, as well as, probably, by an increase in the production of TNF-α, under the influence of which there is a transendothelial migration of lymphocytes into the tissue.

Anti-CD52 Therapy for Multiple Sclerosis: An Update in the COVID Era.

CD52 is a small surface glycoprotein composed of 12 amino acids. CD52 is found mostly on the surface of mature immune cells, such as lymphocytes, monocytes, eosinophils, and dendritic cells, as well as the male genital tract: within the epididymis and on the surface of mature sperm. Low CD52 expression is also found in neutrophils. CD52 function is not fully understood, although experiments with anti-CD52 antibodies have shown that CD52 is essential for lymphocyte transendothelial migration and may contribute to costimulation of CD4+ T cells and T-cell activation and proliferation. Although knowledge about exact CD52 function is still poor, CD52 presence on the surface of a broad spectrum of immune cells makes it a therapeutic target, especially in immunomediated diseases, such as multiple sclerosis. In multiple sclerosis, alemtuzumab is registered for adult patients with the relapsing–remitting form of the disease defined by clinical and imaging features. Despite the high efficacy of the drug, the main issue is its safety. The main adverse effects of alemtuzumab are associated with drug infusion due to cytokine release and cytotoxic effects of antibodies associated with lymphocyte depletion, which leads to immunosuppression, and secondary autoimmunity that may be the effect of excessive B-cell repopulation and cancer. This review presents current knowledge on the drug’s mechanism of action, efficacy and safety data from clinical trials, and real-world observations, including available though scarce data on using alemtuzumab in the COVID era.

Endothelial Protease Activated Receptor 1 (PAR1) Signalling Is Required for Lymphocyte Transmigration across Brain Microvascular Endothelial Cells.

Lymphocyte transendothelial migration (TEM) relies on ICAM-1 engagement on the luminal surface of the endothelial cells (ECs). In blood–brain barrier (BBB) ECs, ICAM-1 triggers TEM signalling, including through JNK MAP kinase and AMP-activated protein kinase (AMPK), which lead to the phosphorylation and internalisation of the adherens junction protein VE-cadherin. In addition to ICAM-1, G protein-coupled receptors (GPCRs) are also required for lymphocytes TEM across BBB ECs. Here, we investigated the role of protease activated GPCRs (PARs) and found a specific role for PAR1 in support of lymphocyte TEM across BBB ECs in vitro. PAR1 requirement for TEM was confirmed using protease inhibitors, specific small molecule and peptide antagonists, function blocking antibodies and siRNA-mediated knockdown. In BBB ECs, PAR1 stimulation led to activation of signalling pathways essential to TEM; notably involving JNK and endothelial nitric oxide synthase (eNOS), with the latter downstream of AMPK. In turn, nitric oxide production through eNOS was essential for TEM by modulating VE-cadherin on Y731. Collectively, our data showed that non-canonical PAR1 activation by a lymphocyte-released serine protease is required for lymphocyte TEM across the BBB in vitro, and that this feeds into previously established ICAM-1-mediated endothelial TEM signalling pathways.

Peculiarities of the expression of the vascular adhesion molecule in acute IA phase pancreatitis

Purpose : to estimate the expression of a molecule of vascular adhesion of PECAM-1 (CD31) at patients with acute pancreatitis of varying severity in ΙА phase diseasedevelopment. Materials and methods . Work has clinical character and is based on the prospective analysis of observation and treatment of 135 patients with acute pancreatitis of various degrees of severity. Patients were divided into three groups, depending on severity of a disease: 1 group — 45 patients with slight acute pancreatitis, the 2 nd group — 45 patients with medium-weight acute pancreatitis, the 3 rd group — 45 patients with heavy acute pancreatitis. Blood sampling for a research was carried out at receipt prior to therapy. Phenotyping of lymphocytes was carried out by method of an indirect immunofl uorescence by means of mouse monoclonal antibodies to the molecules CD31 receptors of lymphocytes. Results . The obtained data demonstrate that the percent of CD31-expressing of lymphocytes depends on weight of acute pancreatitis. At initially severe form of a disease, there was strengthening of process of transendothelial migration of lymphocytes in the center of forming of a necrosis in pancreas tissue. In our research signifi cantly (p <0.001, p1 <0.001, p2 <0.001) the percent of the CD31-expressing lymphocytes to 13.21 increased [12.50; 5.76]. Conclusion . The obtained data demonstrate that there is an interrelation between PECAM1 expression as a marker of intercellular interactions and severity of acute pancreatitis.

In Vivo F-Actin Filament Organization during Lymphocyte Transendothelial and Interstitial Migration Revealed by Intravital Microscopy

SummaryActin is essential for many cellular processes including cell motility. Yet the organization of F-actin filaments during lymphocyte transendothelial migration (TEM) and interstitial migration have not been visualized. Here we report a high-resolution confocal intravital imaging technique with LifeAct-GFP bone marrow reconstituted mice, which allowed visualization of lymphocyte F-actin in vivo. We find that naive lymphocytes preferentially cross high endothelial venules (HEVs) using paracellular rather than the transcellular route. During both modes of transmigration F-actin levels rise at the lymphocyte leading edge as the cell engages the TEM site. Once the lymphocytes breach the endothelium, they briefly reside in HEV pockets before crossing into the parenchyma. During interstitial migration dynamic actin-based protrusions rapidly form and collapse to help drive motility. Using a panel of inhibitors, we established roles for actin regulators and myosin II in lymphocyte TEM. This study provides further insights into lymphocyte TEM and interstitial migration in vivo.

Vimentin is required for normal accumulation of body fat.

Intermediate filaments (nanofilaments) have many functions, especially in response to cellular stress. Mice lacking vimentin (Vim-/-) display phenotypes reflecting reduced levels of cell activation and ability to counteract stress, for example, decreased reactivity of astrocytes after neurotrauma, decreased migration of astrocytes and fibroblasts, attenuated inflammation and fibrosis in lung injury, delayed wound healing, impaired vascular adaptation to nephrectomy, impaired transendothelial migration of lymphocytes and attenuated atherosclerosis. To address the role of vimentin in fat accumulation, we assessed the body weight and fat by dual-energy X-ray absorptiometry (DEXA) in Vim-/- and matched wildtype (WT) mice. While the weight of 1.5-month-old Vim-/- and WT mice was comparable, Vim-/- mice showed decreased body weight at 3.5, 5.5 and 8.5 months (males by 19-22%, females by 18-29%). At 8.5 months, Vim-/- males and females had less body fat compared to WT mice (a decrease by 24%, p < 0.05, and 33%, p < 0.0001, respectively). The body mass index in 8.5 months old Vim-/- mice was lower in males (6.8 vs. 7.8, p < 0.005) and females (6.0 vs. 7.7, p < 0.0001) despite the slightly lower body length of Vim-/- mice. Increased mortality was observed in adult Vim-/- males. We conclude that vimentin is required for the normal accumulation of body fat.

IRGD synergizes with PD-1 knockout immunotherapy by enhancing lymphocyte infiltration in gastric cancer

Poor infiltration of activated lymphocytes into tumors represents a fundamental factor limiting the therapeutic effect of adoptive cell immunotherapy. A tumor-penetrating peptide, iRGD, has been widely used to deliver drugs into tumor tissues. In this study, we demonstrate for the first time that iRGD could also facilitate the infiltration of lymphocytes in both 3D tumor spheroids and several xenograft mouse models. In addition, combining iRGD modification with PD-1 knockout lymphocytes reveals a superior anti-tumor efficiency. Mechanistic studies demonstrate that the binding of iRGD to neuropilin-1 results in tyrosine phosphorylation of the endothelial barrier regulator VE-cadherin, which plays a role in the opening of endothelial cell contacts and the promotion of transendothelial lymphocyte migration. In summary, these results demonstrate that iRGD modification could promote tumor-specific lymphocyte infiltration, and thereby overcome the bottleneck associated with adoptive immune cell therapy in solid tumors.

PO-371 ADAM28 deletion in mice induces CD8 +T cell decrease and impacts the onset of lung metastasis

IntroductionADAM28 is highly overexpressed in NSCL cancer. In addition, intrinsic characteristics of this proteinase argue for considering it as a potential regulator of cellular signalling pathways leading to an inflammatory pulmonary microenvironment and to carcinogenesis. Indeed, ADAM28 bears an active catalytic domain and interacts in a non-proteolytic manner with integrins α4β1 and P-selectin ligands involved in inflammatory cell migration. The aim of this project is to characterise effects of host-ADAM28 on physiological and pathological processes.Material and methodsADAM28 conditional KO mice have been developed in our laboratory. This unique mouse strain provides a precious tool to investigate ADAM28 implication in lung tumour growth and dissemination. Indeed, mice were intravenously injected with Lewis Lung, 4 T1 carcinoma cells and B16K1 melanoma cells. Since ADAM28 is expressed by thymic epithelial cells and implicated in lymphocyte transendothelial migration, FACS analysis has been performed to study lymphocyte subsets implicated in tumour cytotoxicity or in regulation of immune response.Results and discussionsFirst analysis showed that there is no spontaneous phenotype for ADAM28 full knockout animals as they are fertile and do not display any abnormality or defect. Surprisingly, an increased metastasic dissemination in lungs has been observed in KO mice compare to WT littermates. Among infiltrating immune cells implicated in anti-tumour response, only CD8 T cells are decreased in metastatic lungs of ADAM28 KO mice. Interestingly, this decrease is already observed in spleen of tumour free-ADAM28 KO mice. These data suggest a potential role of ADAM28 during lymphocyte maturation process, which could further impact antitumoral functions. Moreover, ex vivo analyses of CD8 T cells demonstrate an increase of PD-L1 at cell surface of CD8 T cells isolated from ADAM28 KO mice. PD-L1 interacts with its PD-1 receptor and their interaction induces CD8 apoptosis and exhaustion. Further investigations are required to demonstrate the link between ADAM28 and PD-L1 and their implication in the decrease of CD8 T cell infiltration.ConclusionThe role of tumour cell-derived ADAM28 as a pro-tumour factor is widely described in the literature. However, our results demonstrate an anti-metastatic function of host derived-ADAM28 suggesting a dual role of ADAM28, depending on the origin of the protease. Based on our results, ADAM28 seems to play a major role in the control of anti-tumour immune response mediated by CD8 T cells.

A novel immunotoxin reveals a new role for CD321 in endothelial cells.

There are currently several antibody therapies that directly target tumors, and antibody-drug conjugates represent a novel moiety as next generation therapeutics. Here, we used a unique screening probe, DT3C, to identify functional antibodies that recognized surface molecules and functional epitopes, and which provided toxin delivery capability. Accordingly, we generated the 90G4 antibody, which induced DT3C-dependent cytotoxicity in endothelial cells. Molecular analysis revealed that 90G4 recognized CD321, a protein localized at tight junctions. Although CD321 plays a pivotal role in inflammation and lymphocyte trans-endothelial migration, little is known about its mechanism of action in endothelial cells. Targeting of CD321 by the 90G4 immunotoxin induced cell death. Moreover, 90G4 immunotoxin caused cytotoxicity primarily in migratory endothelial cells, but not in those forming sheets, suggesting a critical role for CD321 in tumor angiogenesis. We also found that hypoxia triggered redistribution of CD321 to a punctate localization on the basal side of cells, resulting in functional impairment of tight junctions and increased motility. Thus, our findings raise the intriguing possibility that endothelial CD321 presented cellular localization in tight junction as well as multifunctional dynamics in several conditions, leading to illuminate the importance of widely-expressed CD321 as a potential target for antitumor therapy.

Endothelial MAPKs Direct ICAM-1 Signaling to Divergent Inflammatory Functions.

Lymphocyte transendothelial migration (TEM) is critically dependent on intraendothelial signaling triggered by adhesion to ICAM-1. Here we show that endothelial MAPKs ERK, p38, and JNK mediate diapedesis-related and diapedesis-unrelated functions of ICAM-1 in cerebral and dermal microvascular endothelial cells (MVECs). All three MAPKs were activated by ICAM-1 engagement, either through lymphocyte adhesion or Ab-mediated clustering. MAPKs were involved in ICAM-1–dependent expression of TNF-α in cerebral and dermal MVECs, and CXCL8, CCL3, CCL4, VCAM-1, and cyclooxygenase 2 (COX-2) in cerebral MVECs. Endothelial JNK and to a much lesser degree p38 were the principal MAPKs involved in facilitating diapedesis of CD4+ lymphocytes across both types of MVECs, whereas ERK was additionally required for TEM across dermal MVECs. JNK activity was critical for ICAM-1–induced F-actin rearrangements. Furthermore, activation of endothelial ICAM-1/JNK led to phosphorylation of paxillin, its association with VE-cadherin, and internalization of the latter. Importantly ICAM-1–induced phosphorylation of paxillin was required for lymphocyte TEM and converged functionally with VE-cadherin phosphorylation. Taken together we conclude that during lymphocyte TEM, ICAM-1 signaling diverges into pathways regulating lymphocyte diapedesis, and other pathways modulating gene expression thereby contributing to the long-term inflammatory response of the endothelium.

Ultrastructural demonstration of antigen presenting cells in appendix

IntroductionThe human Appendix is a component of gut associated lymphoid tissue (GALT). Lymphoid denritic cells play a pivotal role in antigen presentation in mucosal immune system. A very few studies have demonstrated antigen presenting cells present in it either by cytochemistry or by immunohistochemistry. The aim of the present study is to demonstrate the antigen presenting cells present in normal and infected Appendix by electron microscopy. Materials and methodsTissues obtained from two normal and five infected appendices were processed for electron microscopy and viewed under Philips EM 201C electron microscope. ResultsIn addition to the enterocytes, goblet cells and enteroendocrine cells lining the appendix, M cells were present in the lining epithelium of both normal and infected appendix. Intraepithelial lymphocytes were present in relation to the base and basolateral surface of the M cells. In addition to these cells, dendritic cells (DCs) were present in the epithelium of infected appendix. DCs were present in the subepithelium of normal and infected appendices. In the region of lymphatic follicles, follicular dendritic cells (FDCs) were present which showed ultrastructural heterogeneity. In the interfollicular area, high endothelial venules (HEVs) were present. Transendothelial migration of lymphocytes through the HEVs could be demonstrated. DiscussionThe ultrastructural demonstration of DCs, FDCs, M cells and the transendothelial migration of lymphocytes through HEVs in Appendix confirms that Appendix is truly a secondary lymphoid organ.

Human GPR4 and the Notch signaling pathway in endothelial cell tube formation.

G protein-coupled receptor4 (GPR4) is hypothesized to function as a pH sensor and is important in the regulation of proliferation, migration and angiogenesis of vascular endothelial cells (ECs). Furthermore, the Notch signaling pathway is significant in the regulation of the angiogenic behavior of ECs. However, whether GPR4 regulates angiogenesis via the Notch signaling pathway remains unclear. The present study evaluated the effect of Notch signaling in human GPR4‑induced angiogenesis in HMEC‑1 cells. The results revealed that GPR4 increased Notch1 expression in a time‑dependent manner. In addition, the inhibition of Notch1 expression using small interfering RNA or the Notch receptor inhibitor, γ-secretase inhibitorI, significantly blocked GPR4‑induced HMEC‑1 tube formation and lymphocyte transendothelial migration. Furthermore, the inhibition of Notch1 blocked GPR4‑induced vascular endothelial growth factor and hypoxia-inducible factor1α expression. Thus, it was demonstrated that GPR4 affects ECs by regulating Notch1, a function that may be important for physiological and pathological angiogenesis.

M-sec regulates polarized secretion of inflammatory endothelial chemokines and facilitates CCL2-mediated lymphocyte transendothelial migration.

Activation of endothelial cells by IL-1β triggers the expression of multiple inflammatory cytokines and leukocyte-attracting chemokines. The machineries involved in the secretion of these inducible proteins are poorly understood. With the use of genome-wide transcriptional analysis of inflamed human dermal microvascular endothelial cells, we identified several IL-1β-induced candidate regulators of these machineries and chose to focus our study on TNF-α-induced protein 2 (myeloid-secretory). The silencing of myeloid-secretory did not affect the ability of inflamed endothelial cells to support the adhesion and crawling of effector T lymphocytes. However, the ability of these lymphocytes to complete transendothelial migration across myeloid-secretory-silenced human dermal microvascular endothelial cells was inhibited significantly. These observed effects on lymphocyte transendothelial migration were recovered completely when exogenous promigratory chemokine CXCL12 was overlaid on the endothelial barrier. A polarized secretion assay suggested that the silencing of endothelial myeloid-secretory impairs T effector transendothelial migration by reducing the preferential secretion of endothelial-produced CCL2, a key transendothelial migration-promoting chemokine for these lymphocytes, into the basolateral endothelial compartment. Myeloid-secretory silencing also impaired the preferential secretion of other endothelial-produced inflammatory chemokines, as well as cytokines, such as IL-6 and GM-CSF, into the basolateral endothelial compartment. This is the first evidence of a novel inflammation-inducible machinery that regulates polarized secretion of endothelial CCL2 and other inflammatory chemokines and cytokines into basolateral endothelial compartments and facilitates the ability of endothelial CCL2 to promote T cell transendothelial migration.

Lysophosphatidic acid as a regulator of lymphocyte trafficking in the lymph nodes

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that regulates multiple biological functions, including cell migration, survival, and proliferation, through the binding of specific G-protein coupled receptors. Although LPA is known to enhance immune cell migration, the identity of the LPA-expressing cells and the mechanism by which LPA regulates leukocyte migration in lymphoid tissues have remained unclear. Using imaging mass spectrometry and intravital two-photon microscopy, we examined the localization of LPA in the lymph nodes and determined its role in lymphocyte migration. We found that LPA is produced by the vascular endothelial cells and other stromal cells within the lymph nodes by the action of the ectoenzyme, autotaxin (ATX). We also found that LPA regulates both lymphocyte transendothelial migration across high endothelial venules (HEVs), and interstitial lymphocyte migration, by acting on both stromal cells and lymphocytes through specific cell-surface receptors for LPA. In this review, we focus on recent advances in our understanding of the expression and function of LPA in immune cell trafficking.