Vascular adhesion protein-1 mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells

Vascular adhesion protein-1 (VAP-1) is an amine oxidase and adhesion receptor that is expressed by endothelium in the human liver. The hepatic sinusoids are perfused by blood at low flow rates, and sinusoidal endothelium lacks selectin expression and has low levels of CD31, suggesting that VAP-1 may play a specific role in lymphocyte recruitment to the liver. In support of this we now report the constitutive expression of VAP-1 on human hepatic sinusoidal endothelial cells (HSEC) in vitro and demonstrate that VAP-1 supports adhesion and transmigration of lymphocytes across these cells under physiological shear stress. These are the first studies to report the function of VAP-1 on primary human endothelial cells. Under static conditions lymphocyte adhesion to unstimulated HSEC was dependent on VAP-1 and ICAM-2, whereas adhesion to TNF-alpha-stimulated HSEC was dependent on ICAM-1, VCAM-1, and VAP-1. Under conditions of flow, blocking VAP-1 reduced lymphocyte adhesion to TNF-alpha-treated HSEC by 50% and significantly reduced the proportion of adherent lymphocytes that transmigrated across cytokine or LPS-activated endothelium. In addition, inhibition of the amine oxidase activity of VAP-1 reduced both adhesion and transmigration of lymphocytes to a level similar to that seen with VAP-1 Ab. Thus, VAP-1 can support transendothelial migration as well as adhesion, and both functions are dependent on its enzymatic activity. In the absence of selectins and CD31, VAP-1 may play a specific role in lymphocyte recruitment via hepatic sinusoidal endothelium. Moreover, since VAP-1 is induced on nonhepatic endothelium in response to inflammation, its ability to support lymphocyte transendothelial migration may be an important systemic function of VAP-1.     

Common lymphatic endothelial and vascular endothelial receptor-1 mediates the transmigration of regulatory T cells across human hepatic sinusoidal endothelium

The common lymphatic endothelial and vascular endothelial receptor (CLEVER-1; also known as FEEL-1 and stabilin-1) is a recycling and intracellular trafficking receptor with multifunctional properties. In this study, we demonstrate increased endothelial expression of CLEVER-1/stabilin-1 at sites of leukocyte recruitment to the inflamed human liver including sinusoids, septal vessels, and lymphoid follicles in inflammatory liver disease and tumor-associated vessels in hepatocellular carcinoma. We used primary cultures of human hepatic sinusoidal endothelial cells (HSEC) to demonstrate that CLEVER-1/stabilin-1 expression is enhanced by hepatocyte growth factor but not by classical proinflammatory cytokines. We then showed that CLEVER-1/stabilin-1 supports T cell transendothelial migration across HSEC under conditions of flow with strong preferential activity for CD4 FoxP3(+) regulatory T cells (Tregs). CLEVER-1/stabilin-1 inhibition reduced Treg transendothelial migration by 40% and when combined with blockade of ICAM-1 and vascular adhesion protein-1 (VAP-1) reduced it by >80%. Confocal microscopy demonstrated that 60% of transmigrating Tregs underwent transcellular migration through HSEC via ICAM-1- and VAP-1-rich transcellular pores in close association with CLEVER-1/stabilin-1. Thus, CLEVER-1/stabilin-1 and VAP-1 may provide an organ-specific signal for Treg recruitment to the inflamed liver and to hepatocellular carcinoma.     

Chemokine Transfer by Liver Sinusoidal Endothelial Cells Contributes to the Recruitment of CD4+ T Cells into the Murine Liver

Leukocyte adhesion and transmigration are central features governing immune surveillance and inflammatory reactions in body tissues. Within the liver sinusoids, chemokines initiate the first crucial step of T-cell migration into the hepatic tissue. We studied molecular mechanisms involved in endothelial chemokine supply during hepatic immune surveillance and liver inflammation and their impact on the recruitment of CD4⁺ T cells into the liver. In the murine model of Concanavalin A-induced T cell-mediated hepatitis, we showed that hepatic expression of the inflammatory CXC chemokine ligands (CXCL)9 and CXCL10 strongly increased whereas homeostatic CXCL12 significantly decreased. Consistently, CD4⁺ T cells expressing the CXC chemokine receptor (CXCR)3 accumulated within the inflamed liver tissue. In histology, CXCL9 was associated with liver sinusoidal endothelial cells (LSEC) which represent the first contact site for T-cell immigration into the liver. LSEC actively transferred basolaterally internalized CXCL12, CXCL9 and CXCL10 via clathrin-coated vesicles to CD4⁺ T cells leading to enhanced transmigration of CXCR4⁺ total CD4⁺ T cells and CXCR3⁺ effector/memory CD4⁺ T cells, respectively in vitro. LSEC-expressed CXCR4 mediated CXCL12 transport and blockage of endothelial CXCR4 inhibited CXCL12-dependent CD4⁺ T-cell transmigration. In contrast, CXCR3 was not involved in the endothelial transport of its ligands CXCL9 and CXCL10. The clathrin-specific inhibitor chlorpromazine blocked endothelial chemokine internalization and CD4⁺ T-cell transmigration in vitro as well as migration of CD4⁺ T cells into the inflamed liver in vivo. Moreover, hepatic accumulation of CXCR3⁺ CD4⁺ T cells during T cell-mediated hepatitis was strongly reduced after administration of chlorpromazine. These data demonstrate that LSEC actively provide perivascularly expressed homeostatic and inflammatory chemokines by CXCR4- and clathrin-dependent intracellular transport mechanisms thereby contributing to the hepatic recruitment of CD4⁺ T-cell populations during immune surveillance and liver inflammation.     

Non-specific cellular responses of rainbow trout to Vibrio anguillarum and its extracellular products (ECPs)

We have studied the early inflammatory response induced by Vibrio anguillarum and by its extracellular products (ECPs) in rainbow trout after intraperitoneal injection. The results showed a very similar inflammatory response which included leucopenia, mainly due to lymphopenia, neutrophilia and an increase in the number of circulating monocytes. Melanomacrophages as well as immature leucocytes were frequently observed circulating in the blood of injected rainbow trout. Monocytes often contain phagocytosed bacteria and other, altered cells including erythrocytes and leucocytes. However, neutrophils only occasionally phagocytosed bacteria. Many circulating leucocytes showed important structural alterations. Neutrophils of trout injected with bacteria and ECPs also showed stronger PAS-staining than those of control trout as well as Döhle bodies and swollen granules. A marked vasodilatation was observed in the kidney and spleen which was coincidental with a mobilization of eosinophilic granular cells and an hypertrophy of sinusoidal endothelial cells showing an increase in the number of cytoplasmic granules. An increase in the number of macrophages and melanomacrophages in the kidney and spleen as well as oedema and leucocyte infiltration in the liver and gills were also noted.     

Expression of Cell Adhesion Molecules, their Ligands and Tumour Necrosis Factor α in the Liver of Patients with Metastatic Gastrointestinal Carcinomas

The expression of the following cell adhesion molecules, their β1 and β2 integrin ligands and the cytokine tumour necrosis factor-α (TNF-α) was investigated by light and electron microscope immunohistochemistry in the liver tissue in 20 patients with colorectal and gastric cancer also presenting with liver metastases: intercellular adhesion molecule-1 (ICAM-1), vascular endothelial adhesion molecule-1 (VCAM-1), E-selectin, leucocyte function-associated antigen-1 (LFA-1), macrophage antigen-1 (Mac-1), and very late antigen-4 (VLA-4). We have found a parallel enhancement of the adhesion molecules and of TNF-α in liver sinusoids surrounding metastases. The expression of ICAM-1 was enhanced on sinusoidal cells in all zones of the acinus. VCAM-1 immune reactivity was diffuse but less intensive in the lobule. E-selectin expression was observed in sinusoidal cells attached to metastases. In tumour metastases the expression of ICAM-1, VCAM-1, and E-selectin was visible on the tumour vascular endothelium. Tumour infiltrating host cells sowing positive immunoreactivity for ICAM-1, VCAM-1, LFA-1, Mac-1, and VLA-4 were located mainly at the boundary between liver parenchyma and the metastasis. At the ultrastructural level, ICAM-1-positive immune deposits were observed on the cellular membrane and in some transport vesicles of gastric metastatic cells. Further, the expression of all adhesion molecules was confirmed to sinusoidal endothelial cells and tumour vessels. It is concluded that the enhanced expression of adhesion molecules in liver sinusoids could be a marker for the assessment of the ability of sinusoidal endothelial cells to control the recruitment of leukocytes and monocytes to the metastatic site. They could also direct the adhesion of new circulating tumour cells to sinusoidal endothelium.     

The emerging role of tetraspanin microdomains on endothelial cells

Tetraspanins function as organizers of the cell surface by recruiting specific partner proteins into tetraspanin-enriched microdomains, which regulate processes such as cell adhesion, signalling and intracellular trafficking. Endothelial cells appear to express at least 23 of the 33 human tetraspanins, and a number of recent studies have demonstrated their importance in endothelial cell biology. Tetraspanin CD151 is essential for pathological angiogenesis, which may in part be due to regulation of its main partner proteins, the laminin-binding integrins α3β1, α6β1 and α6β4. CD9 and CD151 are essential for leucocyte recruitment during an inflammatory response, through the formation of pre-assembled nano-platforms containing the adhesion molecules ICAM-1 (intercellular adhesion molecule 1) and VCAM-1 (vascular cell adhesion molecule 1), which ultimately coalesce to form docking structures around captured leucocytes. Tetraspanin CD63 also facilitates leucocyte capture by promoting clustering of the adhesion molecule P-selectin. Finally, Tspan12 is required for blood vessel development in the eye, through regulation of Norrin-induced Frizzled-4 signalling, such that Tspan12 mutations can lead to human disease. Future studies on these and other endothelial tetraspanins are likely to provide further novel insights into angiogenesis and inflammation.     

Vascular cell lines expressing SSAO/VAP-1: a new experimental tool to study its involvement in vascular diseases

Background information. PrAO (primary amine oxidase), also known as SSAO (semicarbazide‐sensitive amine oxidase)/VAP‐1 (vascular adhesion protein‐1), is an enzyme (EC 1.4.3.21) that is highly expressed in blood vessels and participates in many cell processes, including glucose handling or inflammatory leucocyte recruitment. High activity levels of this enzyme are associated with diabetes, atherosclerosis, AD (Alzheimer's disease) or stroke, among others, thus meaning that studies concerning SSAO as a therapeutic target are becoming more frequent. However, the study of this enzyme is difficult, owing to its loss of expression in cell cultures. Results. We have developed an endothelial cell line that stably expresses the human SSAO/VAP‐1 to be used as endothelial cell model for the study of this enzyme. The transfected protein is mainly expressed as a dimer in the membrane of these cells, and we demonstrate its specific localization in the lipid rafts of endothelial cells. The protein shows levels of enzymatic activity and kinetic parameters comparable with those observed in vivo by the same cell type. The transfected SSAO/VAP‐1 is also able to mediate the adhesion of leucocytes to the endothelium, a known function of this protein under inflammatory conditions. This distinctive function is not exerted by the SSAO/VAP‐1 transfected protein in a smooth muscle cell line that expresses 3‐fold higher protein levels. These differences have been widely reported to exist in vivo. Furthermore, using this endothelial cell model, we describe for the first time the involvement of the leucocyte‐adhesion activity of SSAO/VAP‐1 in the Aβ (amyloid β‐peptide)‐mediated pro‐inflammatory effect. Conclusions. The characterization of this new cell line shows the correct behaviour of the transfected protein and endorses the use of these cellular models for the in‐depth study of the currently poorly understood functions of SSAO/VAP‐1 and its involvement in the above‐mentioned pathologies. This cellular model will be also useful for the evaluation of potential compounds that could modulate its activity for therapeutic purposes.     

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.     

Differential inhibition of polymorphonuclear leukocyte recruitment in vivo by dextran sulphate and fucoidan

The selectin-mediated rolling of leukocytes along the endothelial cells is a prerequisite step followed by firm adhesion and extravasation into the inflamed tissue. This initial contact can be suppressed by sulphated polysaccharides. We have studied the effect of sulphated polysaccharides on the ultimate polymorphonuclear leukocyte (PMN) recruitment and plasma leakage in rabbit skin in response to intradermal injection of various inflammatory mediators. PMN infiltration evoked by various PMN chemoattractants (FMLP, C5a desArg, LTB(4) and IL-8) was significantly inhibited after intravenous injection of dextran sulphate (25 mg/kg), heparin (2 x 90 mg/kg) or fucoidan (1 mg/kg). PMN-dependent plasma leakage was equally well reduced by the different sulphated polymers. Vascular permeability induced by histamine or thrombin acting via a PMN-independent mechanism was not reduced. Fucoidan was the only polysaccharide able to suppress IL-1-induced PMN infiltration for 60-70%. Local administration of dextran sulphate had no effect on PMN-dependent plasma leakage. Differential inhibition of PMN recruitment was determined after injection of dextran sulphate or fucoidan depending on the type of insult. Therefore, these results suggest that different adhesion pathways are utilized during PMN recruitment in vivo in response to chemoattractants and IL-1.     

Bradykinin and des-Arg10-kallidin enhance the adhesion of polymorphonuclear leukocytes to extracellular matrix proteins and endothelial cells

Bradykinin-related peptides (kinins) are well known to contribute to leukocyte recruitment to inflammatory foci; however, a role of these universal pro-inflammatory mediators in the first step of this process, i.e. the leukocyte adhesion to endothelial cells, is not well understood. In this work we found that bradykinin and des-Arg10-kallidin enhance the adhesion of polymorphonuclear bloods cells (PMN) to fibrinogen and fibronectin. Also, the PMN adherence to endothelial cells of HMEC-1 line strongly increased after stimulation by kinins, particularly des-Arg10-kallidin, or when PMN were co-stimulated with bradykinin and interleukin-1β. These effects were attenuated after PMN treatment with a specific inhibitor of carboxypeptidases, which convert kinins to their des-Arg metabolites. The kinin peptides were also able to change the Mac-1 integrin expression on the PMN surface. These results suggest a regulatory effect of kinins on leukocyte adhesion to endothelial wall, providing new aspects of the leukocyte infiltration into inflamed tissues.     

Influence of microflow on hepatic sinusoid blood flow and red blood cell deformation

Hepatic sinusoids present complex anatomical structures such as the endothelial sieve pores and the Disse space, which govern the microscopic blood flow in the sinusoids and are associated with structural variations in liver fibrosis and cirrhosis. However, the contributions of the permeability of endothelial and collagen layers and the roughness of hepatocyte microvilli to the features of this microflow remain largely unknown. Here, an immersed boundary method coupled with a lattice Boltzmann method was adopted in an in vitro hepatic sinusoidal model, and flow field and erythrocyte deformation analyses were conducted by introducing three new source terms including permeability of the endothelial layer, resistance of hepatocyte microvilli and collagen layers, and deformation of red blood cells (RBCs). Numerical calculations indicated that alterations in endothelial permeability could significantly affect the flow velocity and flow rate distributions in hepatic sinusoids. Interestingly, a biphasic regulating pattern of shear stress occurred simultaneously on the surface of hepatocytes and the lower side of endothelium, i.e., the shear stress increased with increased thickness of hepatocyte microvilli and collagen layer when the endothelial permeability was high but decreased with the increase of the thickness at low endothelial permeability. Additionally, this specified microflow manipulates typical RBC deformation inside the sinusoid, yielding one-third of the variation of deformable index with varied endothelial permeability. These simulations not only are consistent with experimental measurements using in vitro liver sinusoidal chip but also elaborate the contributions of endothelial and collagen layer permeability and wall roughness. Thus, our results provide a basis for further characterizing this microflow and understanding its effects on cellular migration and deformation in the hepatic sinusoids.     

Alterations in leucocyte trafficking in lupus-prone mice: an examination of the MRL/faslpr mouse

Systemic lupus erythematosus (SLE) is an autoimmune disease involving inappropriate inflammatory responses in a wide range of organs. The recruitment of leucocytes to these sites of inflammation is one of the key events in the development of tissue injury in SLE. However, the mechanisms responsible for this aberrant recruitment are poorly understood. Several studies have demonstrated upregulation of endothelial adhesion molecule expression in tissue biopsies from SLE patients. However, the progression to analysis of the functional roles of these adhesion molecules has entailed the use of animal models of SLE. Much of this work has involved the use of the MRL/faslpr mouse model of systemic autoimmune disease. This mouse develops a systemic inflammatory disease with similarities to human SLE. This review summarizes work by our laboratory and others which have examined alterations in the mechanisms of leucocyte trafficking in the MRL/faslpr mouse. These experiments have revealed upregulation of key adhesion molecules, alterations in leucocyte-endothelial cell interactions and in some cases protective effects of deletion of endothelial adhesion molecules. From analysis of a range of microvasculatures in the MRL/faslpr mouse, it is becoming clear that the roles of specific adhesion molecules vary according to the tissue under analysis. Furthermore, analysis of MRL/faslpr mice with targeted deletions of specific adhesion molecules indicates that their roles in development and progression of disease can vary from having key contributions to the development of disease, to attenuating disease via as yet unidentified mechanisms.     

The liver: a model of organ-specific lymphocyte recruitment

The liver is constantly exposed to gut-derived antigens that enter via the portal vein, and it must modulate immune responses so that harmful pathogens are cleared but necessary food antigens are ignored. The liver contains a large resident and migratory population of lymphocytes and macrophages that provide immune surveillance against foreign antigen. This population of cells can be rapidly expanded in response to infection or injury by recruiting leukocytes from the circulation, a process that is dependent on the ability of lymphocytes to recognise, bind to and migrate across the endothelial cells that line the vasculature. Lymphocytes can enter the liver at several sites: the vascular endothelium in the portal tracts (comprising the hepatic artery, portal vein and bile ductule), the sinusoids (through which the blood percolates past the hepatocytes) or the central hepatic veins (through which the blood exits). The requirements and physical conditions at each site vary and there is evidence that different combinations of adhesion proteins are involved at these different sites. This article discusses the expression and function of adhesion molecules within the liver and demonstrates how specific populations of effector lymphocytes can be selectively recruited to the liver.     

Cell model of inflammation

Chemical and physical stimuli trigger a cutaneous response by first inducing the main epidermal cells, keratinocytes, to produce specific mediators that are responsible for the initiation of skin inflammation. Activation modulates cell communication, namely leucocyte recruitment and blood-to-skin extravasation through the selective barrier of the vascular ECs (endothelial cells). In the present study, we describe an in vitro model which takes into account the various steps of human skin inflammation, from keratinocyte activation to the adhesion of leucocytes to dermal capillary ECs. Human adult keratinocytes were subjected to stress by exposure to UV irradiation or neuropeptides, then the conditioned culture medium was used to mimic the natural micro-environmental conditions for dermal ECs. A relevant in vitro model must include appropriate cells from the skin. This is shown in the present study by the selective reaction of dermal ECs compared with EC lines from distinct origins, in terms of leucocyte recruitment, sensitivity to stress and nature of the stress-induced secreted mediators. This simplified model is suitable for the screening of anti-inflammatory molecules whose activity requires the presence of various skin cells.     

Electron microscopic evidence for a muscle layer in the wall of the terminal hepatic venule in the rat

Summary Electron microscopically smooth muscle cells could be detected in the walls of the terminal hepatic venules of the rat liver. These muscle cells are double layered in the following venous channels, the intercalated veins. It is supposed that these smooth muscle cells play an important role as outlet sphincters of the liver sinusoids. Furthermore, fenestrations could be found in the endothelial lining of terminal hepatic venules.Dedicated to Prof. Dr. Drs. h.c. W. Bargmann on his 70th birthday.Supported by a grant of the Deutsche Forschungsgemeinschaft.     

Bradykinin and des-Arg10-kallidin enhance the adhesion of polymorphonuclear leukocytes to extracellular matrix proteins and endothelial cells

Abstract

Bradykinin-related peptides (kinins) are well known to contribute to leukocyte recruitment to inflammatory foci; however, a role of these universal pro-inflammatory mediators in the first step of this process, i.e. the leukocyte adhesion to endothelial cells, is not well understood. In this work we found that bradykinin and des-Arg¹⁰-kallidin enhance the adhesion of polymorphonuclear bloods cells (PMN) to fibrinogen and fibronectin. Also, the PMN adherence to endothelial cells of HMEC-1 line strongly increased after stimulation by kinins, particularly des-Arg¹⁰-kallidin, or when PMN were co-stimulated with bradykinin and interleukin-1β. These effects were attenuated after PMN treatment with a specific inhibitor of carboxypeptidases, which convert kinins to their des-Arg metabolites. The kinin peptides were also able to change the Mac-1 integrin expression on the PMN surface. These results suggest a regulatory effect of kinins on leukocyte adhesion to endothelial wall, providing new aspects of the leukocyte infiltration into inflamed tissues.     

Chemokine and chemokine receptor interactions provide a mechanism for selective T cell recruitment to specific liver compartments within hepatitis C-infected liver

The role played by chemokines in regulating the selective recruitment of lymphocytes to different tissue compartments in disease is poorly characterized. In hepatitis C infection, inflammation confined to portal areas is associated with a less aggressive course, whereas T cell infiltration of the liver parenchyma is associated with progressive liver injury and cirrhosis. We propose a mechanism to explain how lymphocytes are recruited to hepatic lobules during bursts of necroinflammatory activity in chronic hepatitis C infection. We report here that lymphocytes infiltrating hepatitis C-infected liver express high levels of the chemokine receptors CCR5 and CXCR3. However, whereas the CCR5 ligands macrophage inflammatory protein-1alpha and -1beta were largely confined to vessels within portal tracts, the CXCR3 ligands IFN-inducible protein-10 and monokine-induced by IFN-gamma were selectively up-regulated on sinusoidal endothelium. In vitro, human hepatic sinusoidal endothelial cells secreted IFN-inducible protein-10 and monokine-induced by IFN-gamma in response to stimulation with IFN-gamma in combination with either IL-1 or TNF-alpha. This suggests that intrahepatic Th1 cytokines drive the increased expression of IFN-inducible protein-10 and monokine-induced by IFN-gamma and thereby promote the continuing recruitment of CXCR3-expressing T cells into the hepatic lobule in chronic hepatitis C infection.