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One of the main pathogenic effects of severe dengue virus (DENV) infection is a vascular leak syndrome. There are no available antivirals or specific DENV treatments and without hospital support severe DENV infection can be life-threatening. The cause of the vascular leakage is permeability changes in the endothelial cells lining the vasculature that are brought about by elevated vasoactive cytokine and chemokines induced following DENV infection. The source of these altered cytokine and chemokines is traditionally believed to be from DENV-infected cells such as monocyte/macrophages and dendritic cells. Herein we discuss the evidence for the endothelium as an additional contributor to inflammatory and innate responses during DENV infection which may affect endothelial cell function, in particular the ability to maintain vascular integrity. Furthermore, we hypothesise roles for two factors, sphingosine kinase-1 and microRNAs (miRNAs), with a focus on several candidate miRNAs, which are known to control normal vascular function and inflammatory responses. Both of these factors may be potential therapeutic targets to regulate inflammation of the endothelium during DENV infection.

Neutrophils are a key mediator of the innate immune system and are pivotal in the inflammatory response to infection or tissue damage. Fundamental to the role that neutrophils play in host defence is their interaction with the vascular endothelium. From the initial mobilisation of neutrophils out of the bone marrow to their ultimate transmigration through the vasculature, endothelial cells are a vital component of the inflammatory process. This review focuses on the interactions that take place between neutrophils and the vascular endothelium during the various stages of the inflammatory response. The role of the vascular endothelium in detecting the initial episode of infection or tissue damage is explored and how this ultimately leads to mobilisation of the neutrophils from the bone marrow and into the bloodstream. The recruitment and adhesion of neutrophils to the vascular endothelium is also discussed, with particular emphasis on the various discrete stages of the adhesion cascade and what molecules mediate these steps. In addition, a novel role for the lipid kinase sphingosine kinase in neutrophil adhesion is examined. With the advent of improved imaging techniques and the development of new animal models, this is a dynamic area of research and this review aims to summarise some of the more recent findings., (© 2013.)

Localization of circulating lymphocytes to a site of inflammation is paramount for the development and maintenance of an immune response. In vitro studies using cell lines have previously demonstrated that rolling and adhesion of lymphocytes on endothelium requires CD44 interactions with hyaluronan (HA). To date, whether CD44 has a role in mediating CD4(+)-polarized T-helper 1 (Th1) and Th2 lymphocyte interactions with the endothelium in vivo is yet to be determined. In this study we used intravital microscopy to demonstrate that both Th1 and Th2 lymphocytes use CD44 to roll and adhere to tumor necrosis factor-alpha (TNFalpha)-activated microvasculature. Furthermore, chimeric studies imply that CD44 expression by both the endothelium and lymphocytes is essential for these interactions to occur. HA was also necessary for T cell-endothelial cell interactions in vivo and Th1 and Th2 cells rolled on immobilized HA in vitro via CD44. In vitro, both Th1 and Th2 lymphocytes have increased expression of CD44 and greater binding of fluorescent HA than naive cells. The interactions of Th1 and Th2 cells were entirely dependent upon both P-selectin and CD44 in vivo, but did not appear to be counter ligands in vitro. Taken together, these results suggest that CD44 and HA are key to both Th1 and Th2 lymphocyte interactions with the TNFalpha-activated endothelium and raises the possibility of cooperativity between the P-selectin/PSGL-1 and HA/CD44 pathways for Th1 and Th2 rolling in vivo.

Lymphocyte localization to inflammatory sites is paramount for developing and maintaining an immune response. Rolling is the first step in recruitment, but our knowledge of its mechanisms in Th1 and Th2 CD4(+) lymphocytes is incomplete. Whereas initial studies suggested that Th1 but not Th2 lymphocytes used P-selectin for recruitment, more recent studies have proposed that both subtypes bind selectins. We used intravital microscopy to demonstrate in vivo that polarized Th1 and Th2 lymphocytes both use P-selectin to roll and adhere to cytokine [tumor necrosis factor (TNF)-alpha or interleukin (IL)-4]-activated intestinal microvasculature. The majority of Th1 lymphocyte flux in TNF-alpha- and IL-4-treated animals was P-selectin-dependent. Th1 lymphocytes also interacted with E-selectin to control rolling velocity after TNF-alpha stimulation. Th2 lymphocytes, which make IL-4 but not interferon-gamma, bound P-selectin ex vivo, with more than 95% rolling on P-selectin in vivo. Both Th1 and Th2 lymphocytes regulated rolling velocity by interacting with alpha(4)-integrin. Furthermore, in a model of spontaneous intestinal inflammation (ie, IL-10-deficient mice), both Th1 and Th2 lymphocytes rolled, adhered, and ultimately emigrated into the local microenvironment. These results suggest that both Th1 and Th2 lymphocytes use P-selectin in the initial rolling step in vivo in response to a global activator of the vasculature (TNF), a subtle inducer of P-selectin (IL-4), and pathological inflammation (IL-10-deficient mice).

The acquisition of homing receptors that redirect lymphocyte trafficking to nonlymphoid tissues after antigen encounter is a fundamental aspect of effector T-cell development. Although a role for selectins and their ligands has been well characterized for trafficking of Th1 cells to nonlymphoid sites, mechanisms responsible for Th2 trafficking are not well understood. Using a flow chamber system in which the endothelial interactions of two distinct T-cell populations could be examined simultaneously, we directly compared the requirements for Th1 and Th2 cell tethering and rolling. We found that although Th2 cells expressed significantly lower levels of selectin ligands than Th1 cells, activation of the endothelium by Th2-derived factors induced rolling interactions that were comparable for both Th1 and Th2 populations. Further, in the absence of PSGL-1, no other adhesion molecule could effectively compensate for lack of PSGL-1 to mediate rolling of either Th1 or Th2 cells. Thus, both Th1 and Th2 populations express functional PSGL-1-based selectin ligands for tethering and rolling on activated endothelium, and both effector populations can use PSGL-1 as the dominant scaffold for functional selectin ligand expression.

P-selectin glycoprotein-1 (PSGL-1) supports P-selectin-dependent rolling in vivo and in vitro. However, controversy exists regarding the importance of PSGL-1-dependent and -independent E-selectin rolling. Using antibodies against PSGL-1 and PSGL-1(-/-) mice, we demonstrated abolition of P-selectin-dependent rolling but only partial inhibition of E-selectin-mediated rolling in the cremaster microcirculation following local administration of tumor necrosis factor alpha (TNF-alpha). In vitro studies demonstrated that binding of recombinant mouse E-selectin chimera to PSGL-1(-/-) neutrophils was dramatically decreased in mice treated systemically but not locally with TNF-alpha. Further, PSGL-1 blockade abolished E-selectin-dependent rolling in wild-type mice following systemic TNF-alpha administration but not local TNF-alpha administration. Together, these data support an E-selectin ligand present on PSGL-1(-/-) neutrophils that is down-regulatable upon systemic but not local activation. To determine whether the PSGL-1-independent E-selectin ligand was physiologically important, we used a P- and E-selectin-dependent cutaneous contact hypersensitivity model. Binding studies showed no E-selectin ligand down-regulation in this model. The few cells that rolled on E-selectin ligand following PSGL-1 antibody administration or in PSGL-1 deficiency were sufficient to induce profound contact hypersensitivity. In conclusion, E-selectin mediates PSGL-1-dependent and independent rolling and the latter can be down-regulated by systemic activation and can replace PSGL-1 to support the development of inflammation.

After an ischemic episode, reperfusion causes profound oxidative stress in the vasculature of the afflicted tissue/organ. The dysregulated accumulation of reactive oxygen species (ROS), such as superoxide, has been closely linked to the production and release of proinflammatory mediators, a profound increase in adhesion molecule expression by the vascular endothelium, and infiltration of neutrophils during ischemia-reperfusion (I/R). Superoxide dismutase (SOD) has been shown to protect tissues and organs against I/R-induced injury; however, the drug had to be continuously perfused or kidneys had to be occluded to prevent clearance. We used intravital microscopy, a system that allowed us to visualize neutrophil-endothelial interactions within the mesenteric postcapillary venules of cats subjected to I/R and tested the hypothesis that I/R-induced neutrophil recruitment was inhibited by treatment with SOD2/3. SOD2/3 is a chimeric fusion gene product that contains the mature SOD2 as well as the COOH-terminal "tail" of SOD3 and, unlike the three naturally occurring SODs (SOD1, SOD2, and SOD3), which bear a net negative charge at pH 7.4, SOD2/3 is positively charged and physiologically stable. Our results suggest that not only does SOD2/3 have a much greater efficacy in vivo than the native human SOD2, but its administration prevents I/R-induced neutrophil-endothelial cell interactions and microvascular dysfunction. Moreover, our data support the hypothesis that reactive oxidants mediate I/R-induced injury and that the chimeric recombinant SOD2/3 has the potential to be a therapeutic agent against this debilitating illness.

The rapid and selective accumulation of neutrophils into the lungs is thought to underlie the pulmonary failure that leads to sepsis-related death. In this study we investigated whether neutrophil TLR4 is important in LPS-induced pulmonary neutrophil recruitment by creating chimeric mice (transferring bone marrow between TLR4(+/+) and TLR4(-/-) mice). In TLR4(+/+) mice receiving TLR4(-/-) bone marrow, 6 weeks after transplant TLR4 was absent in all circulating leukocytes as well as in resident macrophages (these mice were termed LeukocyteTLR4(-/-)), and these cells were completely nonresponsive to LPS. In TLR4(-/-) mice receiving TLR4(+/+) bone marrow, endothelial cells but not leukocytes were deficient in TLR4 (EndotheliumTLR4(-/-)). Surprisingly, systemic LPS (0.5 mg/kg) induced a dramatic increase in neutrophil sequestration into the lungs of LeukocyteTLR4(-/-) mice over the first 4 hours. Concomitantly, numbers of circulating leukocytes decreased by 90%. By contrast, EndotheliumTLR4(-/-) mice showed very little increase in neutrophil sequestration in the lungs, suggesting that endothelium rather than leukocyte TLR4 was important. Intravital microscopy of peripheral microcirculation in the cremaster muscle revealed about 30-fold more leukocyte-endothelial cell interactions in LPS-treated EndotheliumTLR4(-/-) mice than in LPS-treated LeukocyteTLR4(-/-) mice. This is consistent with less sequestration of leukocytes into the lungs of EndotheliumTLR4(-/-) mice. In conclusion, our data challenge the view that LPS directly activates neutrophils to trap in lungs and suggest a far more important role than previously appreciated for the endothelial cells.