Activation of endothelial TLR2 by bacterial lipoprotein upregulates proteins specific for the neutrophil response

The vascular endothelium is integrally involved in the host response to infection and in organ failure during acute inflammatory disorders such as sepsis. Gram-negative and Gram-positive bacterial lipoproteins circulate in sepsis and can directly activate the endothelium by binding to endothelial cell (EC) TLR2. In this report, we perform the most comprehensive analysis to date of the immune-related genes regulated after activation of endothelial TLR2 by bacterial di- and triacylated lipopeptides. We found that TLR2 activation specifically induces the expression of the genes IL-6, IL-8, CSF2, CSF3, ICAM1 and SELE by human umbilical vein ECs and human lung microvascular ECs. These proteins participate in neutrophil recruitment, adherence and activation at sites of inflammation. Significantly, our studies demonstrate that TLR2-mediated EC responses are specifically geared towards recruitment, activation, and survival of neutrophils and not mononuclear leukocytes, that ECs do not require priming by other inflammatory stimuli to respond to bacterial lipopeptides and, unlike mononuclear leukocytes, TLR2 agonists do not induce ECs to secrete TNF-α. This study suggests that endothelial TLR2 may be an important regulator of neutrophil trafficking to sites of infection in general, and that direct activation of lung endothelial TLR2 may contribute to acute lung injury during sepsis.     

Neutrophil-derived heparin-binding protein (HBP/CAP37) deposited on endothelium enhances monocyte arrest under flow conditions

In acute inflammation, infiltration of neutrophils often precedes a second phase of monocyte invasion, and data in the literature suggest that neutrophils may directly stimulate mobilization of monocytes via neutrophil granule proteins. In this study, we present a role for neutrophil-derived heparin-binding protein (HBP) in monocyte arrest on endothelium. Adhesion of neutrophils to bovine aorta endothelial cells (ECs) or HUVEC-triggered secretion of HBP and binding of the protein to the EC surface. Blockade of neutrophil adhesion by treatment with a mAb to CD18 greatly reduced accumulation of HBP. In a flow chamber model, immobilized recombinant HBP induced arrest of human monocytes or monocytic Mono Mac 6 (MM6) cells to activated EC or plates coated with recombinant adhesion molecules (E-selectin, P-selectin, VCAM-1). However, immobilized recombinant HBP did not influence arrest of neutrophils or lymphocytes. Treatment of MM6 cells with recombinant HBP evoked a rapid and clear-cut increase in cytosolic free Ca(2+) that was found to be critical for the HBP-induced monocyte arrest inasmuch as pretreatment with the intracellular calcium chelating agent BAPTA-AM abolished the evoked increase in adhesion. Thus, secretion of a neutrophil granule protein, accumulating on the EC surface and promoting arrest of monocytes, could contribute to the recruitment of monocytes at inflammatory loci.     

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system¹. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury^2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes⁴. This method focuses on neutrophil transmigration across human endothelial cells.One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)⁵. Neutrophil isolation has been described visually elsewhere⁶; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-α results in increased E-selectin and IL-8 expression⁷. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature^7,8.The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro^9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi μslide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.     

Role of glucocorticoids in neutrophil and endothelial adhesion molecule expression and function

Glucocorticoids are very effective inhibitors of both the acute and chronic inflammatory response. In this study the hypothesis that glucocorticoids inhibit an early component of the inflammatory response, neutrophil adhesion to endothelium, by down-regulation of adhesion molecules on neutrophils or endothelium was examined. No effect of dexamethasone on neutrophil adhesion to endothelium or of antigen expression by neutrophils or endothelium was found. The mechanism of action of glucocorticoids in the inflammatory response is probably not mediated by alterations in adhesion molecules.     

Activation of endothelial extracellular signal-regulated kinase is essential for neutrophil transmigration: potential involvement of a soluble neutrophil factor in endothelial activation

During an inflammatory response induced by infection or injury, leukocytes traverse the endothelial barrier into the tissue space. Extravasation of leukocytes is a multistep process involving rolling, tethering, firm adhesion to the endothelium, and finally, transendothelial migration, the least characterized step in the process. The resting endothelium is normally impermeable to leukocytes; thus, during inflammation, intracellular signals that modulate endothelial permeability are activated to facilitate the paracellular passage of leukocytes. Using a static in vitro assay of neutrophil transmigration across human umbilical vein endothelium, a panel of inhibitors of intracellular signaling was screened for their ability to inhibit transmigration. PD98059, a specific inhibitor of extracellular signal-regulated kinase (ERK) 1/2 activation, inhibited both transmigration across TNF-alpha-activated endothelium and transmigration induced by the chemoattractant fMLP in a dose-dependent manner. PD98059 did not inhibit neutrophil chemotaxis in the absence of an endothelial barrier nor neutrophil adhesion to the endothelium, suggesting that its effect was on the endothelium, and furthermore, that endothelial ERK activation may be important for transmigration. We demonstrate in this study that endothelial ERK is indeed activated during neutrophil transmigration and that its activation is dependent on the addition of neutrophils to the endothelium. Further characterization showed that the trigger for endothelial ERK activation is a soluble protein of molecular mass approximately 30 kDa released from neutrophils after activation.     

Forging the endothelium during inflammation: pushing at a half-open door?

During an inflammatory response, changes in the adhesive properties of the endothelium occur that enable normally non-adherent blood-borne leukocytes to adhere and subsequently to traverse the endothelium through small gaps at inter-cellular junctions. This review concentrates on the role played by inter-endothelial adhesion molecules during transmigration and the way in which their expression may be regulated during inflammation. We show that the final "open" signals that lead to the formation of clefts between adjacent endothelial cells may be derived from inflamed tissue underlying the endothelium and from activated leukocytes.     

The anti-tumor agent,ingenol-3-angelate (PEP005), promotes the recruitment of cytotoxic neutrophils by activation of vascular endothelial cells in a PKC-δ dependent manner

The modes of action of the novel anti-skin tumor agent ingenol-3-angelate (PEP005) are incompletely understood. Crucially, the cytotoxic functions of neutrophils recruited to the tumor in response to topical application of PEP005 are necessary for effective ablation of the treated lesion. Here, we investigated the hypothesis that the phorbol ester-like properties of PEP005 and its ability to activate PKC could directly activate endothelial cells (EC) so that they support the recruitment of neutrophils. Exposure of EC to PEP005 induced mRNA and/or protein for E-selectin, ICAM-1 and IL-8 in a dose dependent manner, while in a flow based adhesion assay, PEP005 treated EC supported the recruitment of neutrophils at levels comparable to EC stimulated with TNF-alpha. Neutrophil adhesion was inhibited by antibody against E-selectin but not P-selectin. Activation of EC was inhibited by the PKC inhibitor bisindolylmaleimide-1 and confocal immuno-fluorescent studies demonstrated translocation of PKC-delta from the cytosol to the peri-nuclear membrane in response to PEP005. Importantly, the knock down of PKC-delta using siRNA completely abolished neutrophil recruitment to EC subsequently treated with PEP005. Thus, we describe a novel route by which the anti-tumor agent PEP005 regulates the recruitment of cytotoxic leukocytes by directly activating EC in a PKC-delta dependent manner.     

Molecular events during leukocyte diapedesis

The recruitment of leukocytes from the circulation into tissues requires leukocyte migration through the vascular endothelium. The mechanisms by which leukocytes attach and firmly adhere to the endothelial cell surface have been studied in detail. However, much less is known about the last step in this process, the diapedesis of leukocytes through the vascular endothelium. This minireview focuses on the interactions between leukocyte and endothelial cell adhesion molecules that are important during leukocyte extravasation. In the past few years a series of endothelial cell surface and adhesion molecules have been identified that are located at endothelial cell contacts and found to participate in leukocyte diapedesis. These junctional cell adhesion molecules are believed to have an active role in controlling the opening and closure of endothelial cell contacts to allow the passage of leukocytes between adjacent endothelial cells. Alternatively, leukocytes can cross the endothelium at nonjunctional locations, with leukocytes migrating through a single endothelial cell. Further work is clearly needed to understand, in greater detail, the molecular mechanisms that allow leukocytes to cross the endothelium via either the paracellular or the transcellular pathway.     

Leukocyte – endothelial interactions in inflammation

At sites of inflammation, infection or vascular injury local proinflammatory or pathogen-derived stimuli render the luminal vascular endothelial surface attractive for leukocytes. This innate immunity response consists of a well-defined and regulated multi-step cascade involving consecutive steps of adhesive interactions between the leukocytes and the endothelium. During the initial contact with the activated endothelium leukocytes roll along the endothelium via a loose bond which is mediated by selectins. Subsequently, leukocytes are activated by chemokines presented on the luminal endothelial surface, which results in the activation of leukocyte integrins and the firm leukocyte arrest on the endothelium. After their firm adhesion, leukocytes make use of two transmigration processes to pass the endothelial barrier, the transcellular route through the endothelial cell body or the paracellular route through the endothelial junctions. In addition, further circulating cells, such as platelets arrive early at sites of inflammation contributing to both coagulation and to the immune response in parts by facilitating leukocyte–endothelial interactions. Platelets have thereby been implicated in several inflammatory pathologies. This review summarizes the major mechanisms and molecules involved in leukocyte–endothelial and leukocyte-platelet interactions in inflammation.     

Modulation of airway inflammation and bacterial clearance by epithelial cell ICAM-1

Many cell types in the airway express the adhesive glycoprotein for leukocytes intercellular adhesion molecule-1 (ICAM-1) constitutively and/or in response to inflammatory stimuli. In this study, we identified functions of ICAM-1 on airway epithelial cells in defense against infection with Haemophilus influenzae. Initial experiments using a mouse model of airway infection in which the bacterial inoculum was mixed with agar beads that localize inflammation in airways demonstrated that ICAM-1 expression was required for efficient clearance of H. influenzae. Airway epithelial cell ICAM-1 expression required few or no leukocytes, suggesting that epithelial cells could be activated directly by interaction with bacteria. Specific inhibition of ICAM-1 function on epithelial cells by orotracheal injection of blocking antibodies resulted in decreased leukocyte recruitment and H. influenzae clearance in the airway. Inhibition of endothelial cell ICAM-1 resulted in a similar decrease in leukocyte recruitment but did not affect bacterial clearance, indicating that epithelial cell ICAM-1 had an additional contribution to airway defense independent of effects on leukocyte migration. To assess this possibility, we used an in vitro model of neutrophil phagocytosis of bacteria and observed significantly greater engulfment of bacteria by neutrophils adherent to epithelial cells expressing ICAM-1 compared with nonadherent neutrophils. Furthermore, bacterial phagocytosis and killing by neutrophils after interaction with epithelial cells were decreased when a blocking antibody inhibited ICAM-1 function. The results indicate that epithelial cell ICAM-1 participates in neutrophil recruitment into the airway, but its most important role in clearance of H. influenzae may be assistance with neutrophil-dependent bacterial killing.     

VEGF₁₆₄ differentially regulates neutrophil and T cell adhesion through ItgaL- and ItgaM-dependent mechanisms

Leukocyte recruitment to inflamed tissues is the cornerstone of inflammatory responses and the driving force behind the establishment of inflammatory bowel disease, consisting of Crohn's disease and ulcerative colitis. It has been reported that angiogenic cytokines contribute to this inflammatory response that facilitates the chronic nature of disease. We have previously reported (Goebel S, Huang M, Davis WC, Jennings M, Siahaan TJ, Alexander JS, Kevil CG. Am J Physiol Gastrointest Liver Physiol 290: G648-G654, 2006) that vascular endothelial growth factor (VEGF)-A can stimulate neutrophil adhesion to colon microvascular endothelial cells in a β?-integrin (Itgb2)-dependent manner. However, it is not known which of the specific leukocyte integrins are critical for VEGF-A-dependent neutrophil and T cell recruitment. Here we examine the differential importance of either α-integrin (Itga)L or ItgaM in governing neutrophil and T cell adhesion to VEGF-A-activated colonic endothelium. Using an in vitro parallel-plate flow chamber model, we found that genetic deficiency of ItgaM completely blunted neutrophil adhesion to VEGF-A-stimulated endothelium, whereas ItgaL deficiency only partly blocked neutrophil adhesion. Deficiency of ItgaM did significantly decrease neutrophil rolling, whereas deficiency of ItgaL did not. We found that genetic deficiency of either ItgaL or ItgaM did significantly blunt T cell adhesion to VEGF-A-stimulated colon endothelium. We also found that genetic deficiency of these Itgas significantly attenuated T cell rolling behavior. Lastly, we examined whether VEGF-A-mediated leukocyte recruitment occurred through different VEGF receptor (VEGFR) pathways and found that VEGFR2 activation regulates neutrophil recruitment, whereas both VEGFR1 and VEGFR2 modulate T cell recruitment. Together, these data identify differential molecular mechanisms of VEGF-A-mediated leukocyte recruitment.     

Omega-3 Fatty Acids and Inflammation: Novel Interactions Reveal a New Step in Neutrophil Recruitment

Inflammation is a physiological response to tissue trauma or infection, but leukocytes, which are the effector cells of the inflammatory process, have powerful tissue remodelling capabilities. Thus, to ensure their precise localisation, passage of leukocytes from the blood into inflamed tissue is tightly regulated. Recruitment of blood borne neutrophils to the tissue stroma occurs during early inflammation. In this process, peptide agonists of the chemokine family are assumed to provide a chemotactic stimulus capable of supporting the migration of neutrophils across vascular endothelial cells, through the basement membrane of the vessel wall, and out into the tissue stroma. Here, we show that, although an initial chemokine stimulus is essential for the recruitment of flowing neutrophils by endothelial cells stimulated with the inflammatory cytokine tumour necrosis factor-α, transit of the endothelial monolayer is regulated by an additional and downstream stimulus. This signal is supplied by the metabolism of the omega-6-polyunsaturated fatty acid (n-6-PUFA), arachidonic acid, into the eicosanoid prostaglandin-D₂ (PGD₂) by cyclooxygenase (COX) enzymes. This new step in the neutrophil recruitment process was revealed when the dietary n-3-PUFA, eicosapentaenoic acid (EPA), was utilised as an alternative substrate for COX enzymes, leading to the generation of PGD₃. This alternative series eicosanoid inhibited the migration of neutrophils across endothelial cells by antagonising the PGD₂ receptor. Here, we describe a new step in the neutrophil recruitment process that relies upon a lipid-mediated signal to regulate the migration of neutrophils across endothelial cells. PGD₂ signalling is subordinate to the chemokine-mediated activation of neutrophils, but without the sequential delivery of this signal, neutrophils fail to penetrate the endothelial cell monolayer. Importantly, the ability of the dietary n-3-PUFA, EPA, to inhibit this process not only revealed an unsuspected level of regulation in the migration of inflammatory leukocytes, it also contributes to our understanding of the interactions of this bioactive lipid with the inflammatory system. Moreover, it indicates the potential for novel therapeutics that target the inflammatory system with greater affinity and/or specificity than supplementing the diet with n-3-PUFAs.     

JAM-L–mediated leukocyte adhesion to endothelial cells is regulated in cis by α4β1 integrin activation

Junctional adhesion molecules (JAMs) are endothelial and epithelial adhesion molecules involved in the recruitment of circulating leukocytes to inflammatory sites. We show here that JAM-L, a protein related to the JAM family, is restricted to leukocytes and promotes their adhesion to endothelial cells. Cis dimerization of JAM-L is required to engage in heterophilic interactions with its cognate counter-receptor CAR (coxsackie and adenovirus receptor). Interestingly, JAM-L expressed on neutrophils binds CAR independently of integrin activation. However, on resting monocytes and T lymphocytes, which express the integrin VLA-4, JAM-L molecules engage in complexes with VLA-4 and mainly accumulate in their monomeric form. Integrin activation is required for the dissociation of JAM-L–VLA-4 complexes and the accumulation of functional JAM-L dimers, which indicates that the leukocyte integrin VLA-4 controls JAM-L function in cis by controlling its dimerization state. This provides a mechanism through which VLA-4 and JAM-L functions are coordinately regulated, allowing JAM-L to strengthen integrin-dependent adhesion of leukocytes to endothelial cells.     

Transendothelial Migration Enables Subsequent Transmigration of Neutrophils through Underlying Pericytes

During acute inflammation, neutrophil recruitment into extravascular tissue requires neutrophil tethering and rolling on cytokine-activated endothelial cells (ECs), tight adhesion, crawling towards EC junctions and transendothelial migration (TEM). Following TEM, neutrophils must still traverse the subendothelial basement membrane and network of pericytes (PCs). Until recently, the contribution of the PC layer to neutrophil recruitment was largely ignored. Here we analyze human neutrophil interactions with interleukin (IL)-1β-activated human EC monolayers, PC monolayers and EC/PC bilayers in vitro. Compared to EC, PC support much lower levels of neutrophil binding (54.6% vs. 7.1%, respectively) and transmigration (63.7 vs. 8.8%, respectively) despite comparable levels of IL-8 (CXCL8) synthesis and display. Remarkably, EC/PC bilayers support intermediate levels of transmigration (37.7%). Neutrophil adhesion to both cell types is Mac-1-dependent and while ICAM-1 transduction of PCs increases neutrophil adhesion to (41.4%), it does not increase transmigration through PC monolayers. TEM, which increases neutrophil Mac-1 surface expression, concomitantly increases the ability of neutrophils to traverse PCs (19.2%). These data indicate that contributions from both PCs and ECs must be considered in evaluation of microvasculature function in acute inflammation.     

A role for the endothelial glycosaminoglycan hyaluronan in neutrophil recruitment by endothelial cells cultured for prolonged periods

Glycosaminoglycans (GAGs) presented on the surface of endothelial cells (ECs) are believed to influence leukocyte recruitment during inflammation, but their roles remain uncertain. Here we report an in vitro model of prolonged culture of human EC in which the contributions of heparan sulphate (HS) and hyaluronan (HA) to the process of neutrophil recruitment could be studied. Previously, we reported that increasing EC culture duration (up to 20 days) enhanced neutrophil recruitment in response to low dose (1 U/ml) but not high dose (100 U/ml) of tumour necrosis factor-α (TNF). Here we found that HS and HA were present at much higher levels on the surface of day 20 cultures than day 3 cultures. Neutrophil recruitment on both day 3 and day 20 ECs was mediated through CXCR chemokine receptors and interleukin-8 (IL-8). In addition, mRNA levels for TNF receptors, signalling pathway constituents, adhesion receptors, and chemokines involved in neutrophil recruitment were similar for day 3 and day 20 ECs. To test whether the enhanced neutrophil recruitment on day 20 EC was mediated by GAGs, they were removed enzymatically. Removal of HA (but not HS) inhibited neutrophil recruitment, as did antibody blockade of CD44, a counter-receptor for HA on neutrophils. Supernatants from hyaluronidase-treated day 20 ECs were more potent in activating neutrophils than supernatants from untreated EC. Thus, HA has a role in neutrophil recruitment that is revealed in long-term cultures where it increases potency of response to sub-optimal levels of TNF. This effect appears to occur through a dual mechanism involving chemokine presentation and interaction with CD44.     

Role of galectin-3 in leukocyte recruitment in a murine model of lung infection by Streptococcus pneumoniae

Pneumonia can be caused by a variety of pathogens, among which Streptococcus pneumoniae causes one of the most common forms of community-acquired pneumonia. Depending on the invading pathogen, the elements of the immune response triggered will vary. For most pathogens, such as Escherichia coli, neutrophil recruitment involves a well-described family of adhesion molecules, beta(2)-integrins. In the case of streptococcal pneumonia, however, neutrophil recruitment occurs mainly through a beta(2)-integrin-independent pathway. Despite decades of research on this issue, the adhesion molecules involved in neutrophil recruitment during lung infection by S. pneumoniae have not been identified. We have previously shown that galectin-3, a soluble mammalian lectin, can be found in lungs infected by S. pneumoniae, but not by E. coli, and can mediate the adhesion of neutrophils on the endothelial cell layer, implying its role in the recruitment of neutrophils to lungs infected with S. pneumoniae. In this study, using galectin-3 null mice, we report further evidence of the involvement of this soluble lectin in the recruitment of neutrophils to S. pneumonia-infected lungs. Indeed, in the absence of galectin-3, lower numbers of leukocytes, mainly neutrophils, were recruited to the infected lungs during infection by S. pneumoniae. In the case of beta(2)-integrin-dependent recruitment induced by lung infection with E. coli, the number of recruited neutrophils was not reduced. Thus, taken together, our data suggest that galectin-3 plays a role as a soluble adhesion molecule in the recruitment of neutrophils to lungs infected by S. pneumoniae, which induces beta(2)-integrin-independent migration.     

Mechanisms of selective leukocyte recruitment from whole blood on cytokine-activated endothelial cells under flow conditions

Selective recruitment of eosinophils to sites of allergic and parasitic inflammation involves specific adhesion and activation signals expressed on or presented by stimulated endothelial cells. Here we examined leukocyte recruitment on cytokine-activated HUVEC under flow conditions. We perfused whole blood through a flow chamber to examine mechanisms of selective leukocyte recruitment. Although there was substantial recruitment of leukocytes on TNF-alpha-stimulated HUVEC, we found no selective accumulation of any particular leukocyte subpopulations. In contrast, fewer leukocytes were recruited to IL-4-stimulated HUVEC, but the recruitment was selective for eosinophils. We examined the role of adhesion molecules in these interactions and found that eosinophil recruitment was completely blocked with an alpha4 integrin mAb at the shear rates examined. A significant number of neutrophils were also recruited to IL-4-stimulated HUVEC, and these interactions required P-selectin and P-selectin glycoprotein ligand-1. Thus, whole blood perfusion over cytokine-activated endothelium revealed that IL-4-stimulated HUVEC support selective recruitment of eosinophils, whereas TNF-alpha-stimulated HUVEC lack selectivity for any leukocyte subclass.     

Prolonged culture of endothelial cells and deposition of basement membrane modify the recruitment of neutrophils

We tested whether endothelial cell conditioning during prolonged culture and deposition of basement membrane (BM) could modify neutrophil recruitment induced by the inflammatory cytokine, tumour necrosis factor-alpha (TNF). Confluent endothelial cells (EC) from human umbilical veins were cultured for 1 to 20 days and then stimulated with 1, 10 or 100 U/ml of TNF for 4 h. When isolated neutrophils were settled on EC stimulated with the lower doses of TNF, the levels of adhesion and the proportion of adherent cells that transmigrated increased markedly with time of culture. At 100 U/ml TNF, time of culture had little effect on recruitment, but the transmigrated neutrophils moved more slowly under the monolayer in longer-term cultures. The inhibitory effects of function-blocking antibodies against E-selectin and beta2-integrin, and studies in which neutrophils were perfused over short- or long-term cultures, suggested that increased adhesion and migration arose from increased efficiency of neutrophil activation by the EC. Prolonged culture was also associated with deposition of a distinct BM. When fresh EC were seeded on day 20 BM, transmigrated neutrophils moved more slowly under the EC than under control monolayers. Thus, EC change their pro-inflammatory phenotype during prolonged culture, and the deposited basement membrane influences neutrophil migration.     

Tumor blood vessels, a difficult hurdle for infiltrating leukocytes

In spite of a gradual improvement of its therapy, cancer is still a deadly disease for millions of patients. Immunotherapy is one of promising treatment strategies, but several mechanisms counteract the development of a proper anti-tumor immune response and the formation of an effective inflammatory infiltrate. One of the difficult hurdles is the hampered recruitment of leukocytes from the blood into the tumor site. It has been demonstrated that tumor cells evolved mechanisms to escape immunity, based on down regulation of endothelial adhesion molecules. This paper reviews the endothelial cell adhesion molecules that mediate leukocyte recruitment and the regulation of them during tumor development. In addition, an overview will be given of the translational development and clinical application of the specific composition of adhesion molecules on tumor endothelium, in diagnosis and therapy.