Evolving functions of endothelial cells in inflammation

Inflammation is usually analysed from the perspective of tissue-infiltrating leukocytes. Microvascular endothelial cells at a site of inflammation are both active participants in and regulators of inflammatory processes. The properties of endothelial cells change during the transition from acute to chronic inflammation and during the transition from innate to adaptive immunity. Mediators that act on endothelial cells also act on leukocytes and vice versa. Consequently, many anti-inflammatory therapies influence the behaviour of endothelial cells and vascular therapeutics influence inflammation. This Review describes the functions performed by endothelial cells at each stage of the inflammatory process, emphasizing the principal mediators and signalling pathways involved and the therapeutic implications.     

Integrin activation by chemokines: relevance to inflammatory adhesion cascade during T cell migration

The adhesive function of integrins is regulated through cytoplasmic signaling induced by several stimuli, whose process is designated "inside-out signaling". A large number of leukocytes are rapidly recruited to the sites of inflammation where they form an essential component of the response to infection, injury, autoimmune disorders, allergy, tumor invasion, atherosclerosis and so on. The recruitment of leukocytes into tissue is regulated by a sequence of interactions between the circulating leukocytes and the endothelial cells. Leukocyte integrins play a pivotal role in leukocyte adhesion to endothelial cells. During the process, the activation of integrins by various chemoattractants, especially chemokines, is essential for integrin-mediated adhesion in which a signal transduced to the leukocyte converts the functionally inactive integrin to an active adhesive configuration. We have proposed that H-Ras-sensitive activation of phosphoinositide 3 (PI 3)-kinase and subsequent profilin-mediated actin polymerization, can be involved in chemokine-induced integrin-dependent adhesion of T cells. The present review documents the relevance of cytoplasmic signaling and cytoskeletal assembly to integrin-mediated adhesion induced by chemoattractants including chemokines during inflammatory processes. In contrast, various adhesion molecules are known to transduce extracellular information into cytoplasm, which leads to T cell activation and cytokine production from the cells, designated "outside-in signaling". Such a bi-directional "cross-talking" among adhesion molecules and cytokines is most relevant to inflammatory processes by augmenting immune cell migration from circulation into inflamed tissue such as rheumatoid arthritis, tumor invasion, Beh?et's disease and atherosclerosis.     

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.     

Effects of blood products on inflammatory response in endothelial cells in vitro

Background

Transfusing blood products may induce inflammatory reactions within the vascular compartment potentially leading to a systemic inflammatory response. Experiments were designed to assess the inflammatory potential of different blood products in an endothelial cell-based in vitro model and to compare baseline levels of potentially activating substances in transfusion products.

Methods

The inflammatory response from pre-activated (endotoxin-stimulated) and non-activated endothelial cells as well as neutrophil endothelial transmigration in response to packed red blood cells (PRBC), platelet concentrates (PC) and fresh frozen plasma (FFP) was determined. Baseline inflammatory mediator and lipid concentrations in blood products were evaluated.

Results

Following incubation with all blood products, an increased inflammatory mediator release from endothelial cells was observed. Platelet concentrates, and to a lesser extent also FFP, caused the most pronounced response, which was accentuated in already pre-stimulated endothelial cells. Inflammatory response of endothelial cells as well as blood product-induced migration of neutrophils through the endothelium was in good agreement with the lipid content of the according blood product.

Conclusion

Within the group of different blood transfusion products both PC and FFP have a high inflammatory potential with regard to activation of endothelial cells. Inflammation upon blood product exposure is strongly accentuated when endothelial cells are pre-injured. High lipid contents in the respective blood products goes along with an accentuated inflammatory reaction from endothelial cells.     

CD40/CD40L interaction induces E-selectin dependent leukocyte adhesion to human endothelial cells and inhibits endothelial cell migration

Background

CD40 is a receptor expressed on a wide range of cells such as leukocytes and endothelial cells (EC). As a member of the tumor necrosis factor (TNF) superfamily the activation of CD40 by CD40-ligand (CD40L) plays a crucial role for the development and progression of a variety of inflammatory processes including atherosclerosis. The aim of the present study was to investigate the effect of CD40/CD40L interaction on leukocyte adhesion to the endothelium and on endothelial cell migration.

Methods and results

Human umbilical vein endothelial cells (HUVEC) were stimulated with either stable transfectants of mouse myeloma cells expressing the CD40L or wild type cells (4 h). Subsequently adhesion of leukocytes expressing Sialyl Lewis X, the counterpart for E-selectin (HL60 cells), was measured under shear stress (2–2.6 dyne/cm²) using a flow chamber adhesion assay. Stimulation of CD40 led to a significant increase of E-selectin dependent adhesion of leukocytes to the endothelium. Incubation of cells with either the CD40L blocking antibody TRAP-1 or the E-selectin blocking antibody BBA2 during CD40 stimulation completely abolished adhesion of leukocytes to HUVEC. Similar results were found in human cardiac microvasculature endothelial cells (HCMEC). In contrast stimulation of CD40 had no effect on adhesion of l-selectin expressing NALM6-L cells. Furthermore, CD40/CD40L interaction abrogated VEGF-induced migration of HUVEC compared to non-stimulated controls. In comparison experiments, stimulation of endothelial cells with VEGF led to a significant phosphorylation of ERK1/2, Akt, and eNOS. Stimulation of endothelial CD40 had no effect on VEGF-induced phosphorylation of ERK1/2. However, VEGF-induced activation of Akt and eNOS was reduced to baseline levels when endothelial CD40 was stimulated.

Conclusion

CD40/CD40L interaction induces E-selectin dependent adhesion of leukocytes to human endothelial cells and reduces endothelial cell migration by inhibiting the Akt/eNOS signaling pathway.     

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.     

Inflammation and neovascularization in diabetic atherosclerosis

Diabetes mellitus, the major cardiovascular risk factor, accentuates the inflammation and neovascularization processes leading to enhanced progression of atherosclerotic complications. Inflammation in diabetes mellitus is the key initiator of atherosclerotic process, which results in acute coronary events. Atherosclerosis evolves from the endothelial cell dysfunction and succeeding entry of hemodynamically derived leukocytes by migration, activation and production of lipid gruel leading to atheromatous plaque progression and subsequent regression. Diabetic plaque progression is associated with increased neovascularization, which is a nature's compliment in the sustenance of plaque growth by its nutrient supply. Neovessels may act as conduit for lipid debridment and alternative channel for inflammatory process. In addition, neovascularization induces intra-plaque hemorrhage due to the fragility of the neovessels and associated inflammation, resulting in plaque instability. The intra-plaque hemorrhage is a detrimental base, which begets the progress of atheroma by inducing oxidative stress and endothelial dysfunction. Intra-plaque hemorrhage is increased in diabetes with an associated increase in hemoglobin-haptoglobin complex (Hb-Hp2-2), which further induces oxidative stress and endothelial cell dysfunction. We conclude that inflammation and neovascularization of the plaque may act as major mechanism augmenting plaque instability in diabetes mellitus.     

The heterogenic properties of monocytes/macrophages and neutrophils in inflammatory response in diabetes

Inflammation is a complicated biological process in response to harmful stimuli, which involves the cooperation of immune system and vascular system. Upon pathogen invasion or tissue injury, resident innate immune cells such as macrophages and dendritic cells are activated and release inflammatory mediators, which result in the vasodilation and recruitment of leukocytes, mainly monocytes and neutrophils. As two of the most important inflammation-mediating immune cells, macrophages and neutrophils have long been regarded to have a pro-inflammatory effect. However, increasing evidences suggest the role of macrophage and neutrophil in inflammation is more complicated and diversified than we thought. Differently activated macrophages and neutrophils lead to diverse even opposite activities. Precise understanding of the role of different subpopulations is critical to achieve the effective treatment for inflammatory diseases. In this review, we discuss the two potentially distinct activation routes of macrophages and neutrophils in obesity and diabetes.     

Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease

Inflammation occurs as a result of exposure of tissues and organs to harmful stimuli such as microbial pathogens, irritants, or toxic cellular components. The primary physical manifestations of inflammation are redness, swelling, heat, pain, and loss of function to the affected area. These processes involve the major cells of the immune system, including monocytes, macrophages, neutrophils, basophils, dendritic cells, mast cells, T-cells, and B-cells. However, examination of a range of inflammatory lesions demonstrates the presence of specific leukocytes in any given lesion. That is, the inflammatory process is regulated in such a way as to ensure that the appropriate leukocytes are recruited. These events are in turn controlled by a host of extracellular molecular regulators, including members of the cytokine and chemokine families that mediate both immune cell recruitment and complex intracellular signalling control mechanisms that characterise inflammation. This review will focus on the role of the main cytokines, chemokines, and their receptors in the pathophysiology of auto-inflammatory disorders, pro-inflammatory disorders, and neurological disorders involving inflammation.     

Interactions between leukocytes and endothelial cells in gout: lessons from a self-limiting inflammatory response

Interactions with endothelium are necessary for leukocytes to pass from the blood into extravascular tissues, and such interactions are facilitated in inflammation by the coordinated expression of endothelial adhesion molecules and chemoattractants. Although the general mechanisms and intracellular pathways of endothelial activation are now fairly well characterised in vitro, relatively little detailed information exists on how endothelial activation changes during the course of inflammatory responses and how such change influences the amount of leukocyte recruitment and the types of leukocytes recruited. Having developed a radiolabelled-antibody-uptake technique for quantifying the expression of endothelial adhesion molecules in relation to leukocyte trafficking, we have analysed the acute, self-limiting inflammatory response to injection of monosodium urate (MSU) crystals. Our studies have supported the view that endothelial activation is closely paralleled by leukocyte recruitment at the onset of the response and have highlighted separate vascular and extravascular stages of downregulation. More recent studies addressing the extravascular contribution to downregulation point to an important role for monocyte-macrophage differentiation in limiting further endothelial activation as a consequence of phagocytosis of MSU crystals.     

Role of superoxide anion radicals in microvascular permeability and leukocyte behaviour

Inflammation is associated with the accumulation and activation of phagocytic cells, such as polymorphonuclear leukocytes, and with the subsequent release and generation of a group of activated oxygen species, some of which are free radicals. These studies were carried out to assess the influence of enzymatically generated free radicals on both microvascular permeability and leukocyte adhesion. The extravasation of fluorescein-labelled dextran mean molecular weight (MW) 150 000 was used to assess microvascular permeability and methodology was developed to measure in vivo leukocyte endothelial interactions. Enzymatically generated superoxide anion radical (O-.2) was associated with an increase in macromolecular extravasation (seen primarily from postcapillary venules) and an increase in leukocyte adhesion. Macromolecular extravasation was found to be dependent on the generation of a hydroxyl radical related interaction while leukocyte adhesion was dependent on the presence of O-.2. It is suggested that the permeability alterations and increased polymorphonuclear leukocyte adhesion seen during inflammation may be partially related to the release of free radicals from inflammatory cells.     

Role of reactive oxygen and nitrogen species in the vascular responses to inflammation

Inflammation is a complex and potentially life-threatening condition that involves the participation of a variety of chemical mediators, signaling pathways, and cell types. The microcirculation, which is critical for the initiation and perpetuation of an inflammatory response, exhibits several characteristic functional and structural changes in response to inflammation. These include vasomotor dysfunction (impaired vessel dilation and constriction), the adhesion and transendothelial migration of leukocytes, endothelial barrier dysfunction (increased vascular permeability), blood vessel proliferation (angiogenesis), and enhanced thrombus formation. These diverse responses of the microvasculature largely reflect the endothelial cell dysfunction that accompanies inflammation and the central role of these cells in modulating processes as varied as blood flow regulation, angiogenesis, and thrombogenesis. The importance of endothelial cells in inflammation-induced vascular dysfunction is also predicated on the ability of these cells to produce and respond to reactive oxygen and nitrogen species. Inflammation seems to upset the balance between nitric oxide and superoxide within (and surrounding) endothelial cells, which is necessary for normal vessel function. This review is focused on defining the molecular targets in the vessel wall that interact with reactive oxygen species and nitric oxide to produce the characteristic functional and structural changes that occur in response to inflammation. This analysis of the literature is consistent with the view that reactive oxygen and nitrogen species contribute significantly to the diverse vascular responses in inflammation and supports efforts that are directed at targeting these highly reactive species to maintain normal vascular health in pathological conditions that are associated with acute or chronic inflammation.     

Protein tyrosine kinases in neutrophil activation and recruitment

Migration of leukocytes into tissue is a key element of innate and adaptive immunity. The first contact of leukocytes with endothelial cells is mediated by engagement of selectins with their counter-receptors which results in leukocyte rolling. During rolling, leukocytes collect different inflammatory signals that activate intracellular signaling pathways. Integration of these signals induces leukocyte activation, firm arrest, post-adhesion strengthening, intravascular crawling, and transmigration. In neutrophils, like in T-cells and platelets, both G-protein-coupled receptor-dependent and -independent activation pathways exist that lead to integrin activation. Accumulating evidence suggests that different protein tyrosine kinases play key roles in signal transduction pathways regulating neutrophil activation and recruitment to inflammatory sites. This review focuses on the role of protein tyrosine kinases of the Src, Syk, and Tec families for neutrophil activation and recruitment.     

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.     

Junctional adhesion molecule-C regulates the early influx of leukocytes into tissues during inflammation

Leukocyte recruitment from blood to inflammatory sites occurs in a multistep process that involves discrete molecular interactions between circulating and endothelial cells. Junctional adhesion molecule (JAM)-C is expressed at different levels on endothelial cells of lymphoid organs and peripheral tissues and has been proposed to regulate neutrophil migration by its interaction with the leukocyte integrin Mac-1. In the present study, we show that the accumulation of leukocytes in alveoli during acute pulmonary inflammation in mice is partially blocked using neutralizing Abs against JAM-C. To confirm the function of JAM-C in regulating leukocyte migration in vivo, we then generated a strain of transgenic mice overexpressing JAM-C under the control of the endothelial specific promotor Tie2. The transgenic animals accumulate more leukocytes to inflammatory sites compared with littermate control mice. Intravital microscopy shows that this is the result of increased leukocyte adhesion and transmigration, whereas rolling of leukocytes is not significantly affected in transgenic mice compared with littermates. Thus, JAM-C participates in the later steps of the leukoendothelial adhesion cascade.