Changes in the pattern of plasma extracellular vesicles after severe trauma.

BACKGROUND: Extracellular vesicles (EV) released into the circulation after traumatic injury may influence complications. We thus evaluated the numbers of EV in plasma over 28 days after trauma and evaluated their pro-coagulant and inflammatory effects. METHODS AND FINDINGS: 37 patients suffering trauma with an injury severity score >15 were studied along with 24 healthy controls. Plasma samples were isolated by double centrifugation (2000g 20min; 13000g 2min) from blood collected from within an hour up to 28 days after injury. Plasma EV were counted and sized using nanoparticle tracking analysis (NTA); counts and cellular origins were also determined by flow cytometry (FC) using cell-specific markers. Functional effects were tested in a procoagulant phospholipid assay and in flow-based, leukocyte adhesion assay after endothelial cells (EC) were treated with EV. We found that EV concentrations measured by NTA were significantly increased in trauma patients compared to healthy controls, and remained elevated over days. In addition, or FC showed that patients with trauma had higher numbers of EV derived from platelets (CD41+), leukocytes (CD45+) and endothelial EC (CD144+). The increases were evident throughout the 28-day follow-up. However, the FC count represented <1% of the count detected by NTA, and only 1-2% of EV identified using NTA had a diameter >400nm. The procoagulant phospholipid activity assay showed that patient plasma accelerated coagulation on day 1 and day 3 after trauma, with coagulation times correlated with EV counts. Furthermore, treatment of EC for 24 hours with plasma containing EV tended to increase the recruitment of peripheral flowing blood mononuclear cells. CONCLUSIONS: EV counted by FC represent a small sub-population of the total load detected by NTA. Both methods however indicate a significant increase in plasma EV after severe traumatic injury that have pro-coagulant and pro-inflammatory effects that may influence outcomes.

Monocyte Subsets Coregulate Inflammatory Responses by Integrated Signaling through TNF and IL-6 at the Endothelial Cell Interface.

Two major monocyte subsets, CD14+CD16- (classical) and CD14+/dimCD16+ (nonclassical/intermediate), have been described. Each has different functions ascribed in its interactions with vascular endothelial cells (EC), including migration and promoting inflammation. Although monocyte subpopulations have been studied in isolated systems, their influence on EC and on the course of inflammation has been ignored. In this study, using unstimulated or cytokine-activated EC, we observed significant differences in the recruitment, migration, and reverse migration of human monocyte subsets. Associated with this, and based on their patterns of cytokine secretion, there was a difference in their capacity to activate EC and support the secondary recruitment of flowing neutrophils. High levels of TNF were detected in cocultures with nonclassical/intermediate monocytes, the blockade of which significantly reduced neutrophil recruitment. In contrast, classical monocytes secreted high levels of IL-6, the blockade of which resulted in increased neutrophil recruitment. When cocultures contained both monocyte subsets, or when conditioned supernatant from classical monocytes cocultures (IL-6hi) was added to nonclassical/intermediate monocyte cocultures (TNFhi), the activating effects of TNF were dramatically reduced, implying that when present, the anti-inflammatory activities of IL-6 were dominant over the proinflammatory activities of TNF. These changes in neutrophil recruitment could be explained by regulation of E-selectin on the cocultured EC. This study suggests that recruited human monocyte subsets trigger a regulatory pathway of cytokine-mediated signaling at the EC interface, and we propose that this is a mechanism for limiting the phlogistic activity of newly recruited monocytes.

Bone Morphogenetic Protein 9 Enhances Lipopolysaccharide-Induced Leukocyte Recruitment to the Vascular Endothelium.

Bone morphogenetic protein (BMP)9 is a circulating growth factor that is part of the TGF-ß superfamily and is an essential regulator of vascular endothelial homeostasis. Previous studies have suggested a role for BMP9 signaling in leukocyte recruitment to the endothelium, but the directionality of this effect and underlying mechanisms have not been elucidated. In this study, we report that BMP9 upregulates TLR4 expression in human endothelial cells and that BMP9 pretreatment synergistically increases human neutrophil recruitment to LPS-stimulated human endothelial monolayers in an in vitro flow adhesion assay. BMP9 alone did not induce neutrophil recruitment to the endothelium. We also show that E-selectin and VCAM-1, but not ICAM-1, are upregulated in response to BMP9 in LPS-stimulated human endothelial cells. Small interfering RNA knockdown of activin receptor-like kinase 1 inhibited the BMP9-induced expression of TLR4 and VCAM-1 and inhibited BMP9-induced human neutrophil recruitment to LPS-stimulated human endothelial cells. BMP9 treatment also increased leukocyte recruitment within the pulmonary circulation in a mouse acute endotoxemia model. These results demonstrate that although BMP9 alone does not influence leukocyte recruitment, it primes the vascular endothelium to mount a more intense response when challenged with LPS through an increase in TLR4, E-selectin, and VCAM-1 and ultimately through enhanced leukocyte recruitment.

The role of platelets in the recruitment of leukocytes during vascular disease.

Besides their role in the formation of thrombus during haemostasis, it is becoming clear that platelets contribute to a number of other processes within the vasculature. Indeed, the integrated function of the thrombotic and inflammatory systems, which results in platelet-mediated recruitment of leukocytes, is now considered to be of great importance in the propagation, progression and pathogenesis of atherosclerotic disease of the arteries. There are three scenarios by which platelets can interact with leukocytes: (1) during haemostasis, when platelets adhere to and are activated on sub-endothelial matrix proteins exposed by vascular damage and then recruit leukocytes to a growing thrombus. (2) Platelets adhere to and are activated on stimulated endothelial cells and then bridge blood borne leukocytes to the vessel wall and. (3) Adhesion between platelets and leukocytes occurs in the blood leading to formation of heterotypic aggregates prior to contact with endothelial cells. In the following review we will not discuss leukocyte recruitment during haemostasis, as this represents a physiological response to tissue trauma that can progress, at least in its early stages, in the absence of inflammation. Rather we will deal with scenarios 2 and 3, as these pathways of platelet-leukocyte interactions are important during inflammation and in chronic inflammatory diseases such as atherosclerosis. Indeed, these interactions mean that leukocytes possess means of adhesion to the vessel wall under conditions that may not normally be permissive of leukocyte-endothelial cell adhesion, meaning that the disease process may be able to bypass the regulatory pathways which would ordinarily moderate the inflammatory response.

Homeostatic regulation of T cell trafficking by a B cell-derived peptide is impaired in autoimmune and chronic inflammatory disease.

During an inflammatory response, lymphocyte recruitment into tissue must be tightly controlled because dysregulated trafficking contributes to the pathogenesis of chronic disease. Here we show that during inflammation and in response to adiponectin, B cells tonically inhibit T cell trafficking by secreting a peptide (PEPITEM) proteolytically derived from 14.3.3 zeta delta (14.3.3.ζδ) protein. PEPITEM binds cadherin-15 on endothelial cells, promoting synthesis and release of sphingosine-1 phosphate, which inhibits trafficking of T cells without affecting recruitment of other leukocytes. Expression of adiponectin receptors on B cells and adiponectin-induced PEPITEM secretion wanes with age, implying immune senescence of the pathway. Additionally, these changes are evident in individuals with type 1 diabetes or rheumatoid arthritis, and circulating PEPITEM in patient serum is reduced compared to that of healthy age-matched donors. In both diseases, tonic inhibition of T cell trafficking across inflamed endothelium is lost. Control of patient T cell trafficking is re-established by treatment with exogenous PEPITEM. Moreover, in animal models of peritonitis, hepatic ischemia-reperfusion injury, Salmonella infection, uveitis and Sjögren's syndrome, PEPITEM reduced T cell recruitment into inflamed tissues.

Pharmacology and therapeutics of omega-3 polyunsaturated fatty acids in chronic inflammatory disease.

Omega-3 (n-3) polyunsaturated fatty acids (n-3 PUFAs) have well documented anti-inflammatory properties, and consequently therapeutic potential in chronic inflammatory diseases. Here we discuss the effects of n-3 PUFAs on various inflammatory pathways and how this leads to alterations in the function of inflammatory cells, most importantly endothelial cells and leukocytes. Strong evidence indicates n-3 PUFAs are beneficial as a dietary supplement in certain diseases such as rheumatoid arthritis; however for other conditions such as asthma, the data are less robust. A clearer understanding of the pharmacology of n-3 PUFAs will help to establish targets to modulate chronic inflammatory diseases.

Adhesion of tumor cells to matrices and endothelium.

Adhesion of tumor cells to matrix components and endothelial cells is essential for tumor metastasis. Investigation of the adhesion molecules required and the signals which induce tumor cell adhesion and migration are crucial in order to increase our understanding of this process. This chapter describes protocols which may be used to study tumor cell adhesion to purified matrix elements and tissue sections. It also details methods used to investigate cell adhesion to endothelial cells, both under static and flow conditions. In addition, there is a section detailing the use of endothelial cell cultures on three-dimensional collagen gels which are useful when studying adhesion to endothelial cells and onward invasion through a protein matrix.

Prostaglandin D2 regulates CD4+ memory T cell trafficking across blood vascular endothelium and primes these cells for clearance across lymphatic endothelium.

Memory lymphocytes support inflammatory and immune responses. To do this, they enter tissue via blood vascular endothelial cells (BVEC) and leave tissue via lymphatic vascular endothelial cells (LVEC). In this study, we describe a hierarchy of signals, including novel regulatory steps, which direct the sequential migration of human T cells across the blood and the lymphatic EC. Cytokine-stimulated (TNF and IFN) human BVEC preferentially recruited memory T cells from purified PBL. Lymphocyte recruitment from flow could be blocked using a function-neutralizing Ab against CXCR3. However, a receptor antagonist directed against the PGD(2) receptor DP2 (formerly chemoattractant receptor-homologous molecule expressed on Th2 cells) inhibited transendothelial migration, demonstrating that the sequential delivery of the chemokine and prostanoid signals was required for efficient lymphocyte recruitment. CD4(+) T cells recruited by BVEC migrated with significantly greater efficiency across a second barrier of human LVEC, an effect reproduced by the addition of exogenous PGD(2) to nonmigrated cells. Migration across BVEC or exogenous PGD(2) modified the function, but not the expression, of CCR7, so that chemotaxis toward CCL21 was significantly enhanced. Thus, chemokines may not regulate all stages of lymphocyte migration during inflammation, and paradigms describing their trafficking may need to account for the role of PGD(2).

Docosahexaenoic acid inhibits the adhesion of flowing neutrophils to cytokine stimulated human umbilical vein endothelial cells.

The (n-3) PUFA, DHA, is widely thought to posses the ability to modulate the inflammatory response. However, its modes of interaction with inflammatory cells are poorly understood. In particular, there are limited data on the interactions of DHA with vascular endothelium, the cells that regulate the traffic of leukocytes from the blood into inflamed tissue. Using human umbilical vein endothelial cells (EC) cultured in a flow-based adhesion assay and activated with TNFα, we tested whether supplementing human umbilical vein EC with physiologically achievable concentrations of DHA would inhibit the recruitment of flowing neutrophils. DHA caused a dose-dependent reduction in neutrophil recruitment to the EC surface, although cells that became adherent were activated and could migrate across the human umbilical vein EC monolayer normally. Using EPA as an alternative supplement had no effect on the levels of neutrophil adhesion in this assay. Analysis of adhesion receptor expression by qPCR demonstrated that DHA did not alter the transcriptional activity of human umbilical vein EC. However, DHA did significantly reduce E-selectin expression at the human umbilical vein EC surface without altering the total cellular pool of this adhesion receptor. Thus, we have identified a novel mechanism by which DHA alters the trafficking of leukocytes during inflammation and demonstrate that this involves disruption of intracellular transport mechanisms used to present adhesion molecules on the surface of cytokine-stimulated EC.

Endovascular aneurysm repair reverses the increased titer and the inflammatory activity of interleukin-1α in the serum of patients with abdominal aortic aneurysm.

OBJECTIVE: To examine serum cytokine/chemokine profiles before and 6 months after endovascular repair (EVAR) of abdominal aortic aneurysm (AAA) and to determine whether they correlate with serum inflammatory activity using an in vitro model of leukocyte recruitment. METHODS: Serum IL-1-α, IL-1ß, IL-4, IL-6, IL-8, IL-10, IFN-γ, IP-10, MCP-1, TNF-α, and TNF-ß were measured using a cytometry-based immunoassay. To test patient serum for direct inflammatory activity, human endothelial cells (EC) were stimulated with 30% patient serum for 24 hours. To test patient serum for the ability to prime EC for inflammatory responses, EC were incubated with 30% patient serum for 24 hours, followed by stimulation with low-dose (5 U/mL) TNF for 4 hours. Under both regimens of stimulation, the degree of EC activation was assessed by assaying neutrophil recruitment in a flow-based model. RESULTS: Only IL-1α (67.9 ± 10.4 pg/mL vs 41.9 ± 7.4 pg/mL) and IL-8 (51.5 ± 5.1 vs 32.6 ± 4.7 pg/mL) changed significantly after surgery. Patient serum alone was unable to activate EC. However, serum from both time points could prime EC responses to low-dose TNF. Thus, after priming with preoperative serum, EC stimulated with TNF could recruit 76.7 ± 12.0 neutrophils/mm(2) into the subendothelial cell space. Post-EVAR serum was significantly less effective (44.4 ± 10.2 neutrophils/mm(2)). This reduction in neutrophil recruitment correlated with reduced IL-1α in post-EVAR serum. The addition of a neutralizing antibody against IL-1α to pre-EVAR serum inhibited EC priming and neutrophil recruitment, strongly implying that this cytokine was the priming agent. CONCLUSION: EVAR reduces serum IL-1α and its inflammatory activity in patient serum. IL-1α is, therefore, implicated in the molecular pathology of AAAs and may have potential as a clinically useful biomarker.

Endothelial cell-borne platelet bridges selectively recruit monocytes in human and mouse models of vascular inflammation.

AIMS: Cells of the monocyte lineage are the most abundant inflammatory cells found in atherosclerotic lesions. Dominance of the inflammatory infiltrate by monocytes indicates that there is a disease-driven mechanism supporting their selective recruitment. Previous studies have demonstrated that interactions between endothelial cells (ECs) and platelets may promote monocyte recruitment. In this study, we sought to expand on this knowledge using a complex coculture model of the diseased vessel wall. METHODS AND RESULTS: Using primary human cells in an in vitro flow-based adhesion assay, we found that secretory arterial smooth muscle cells (SMCs), cocultured with ECs, promote preferential recruitment of monocytes from blood in a TGF-ß1-dependent manner. Approximately 85% of leucocytes recruited to the endothelium were CD14(+). Formation of adhesive platelet bridges on ECs was essential for monocyte recruitment as platelet removal or inhibition of adhesion to the ECs abolished monocyte recruitment. Monocytes were recruited from flow by platelet P-selectin and activated by EC-derived CC chemokine ligand 2 (CCL2), although the presentation of CCL2 to adherent monocytes was dependent upon platelet activation and release of CXC chemokine ligand 4 (CXCL4). In an intravital model of TGF-ß1-driven vascular inflammation in mice, platelets were also necessary for efficient leucocyte recruitment to vessels of the microcirculation in the cremaster muscle. CONCLUSIONS: In this study, we have demonstrated that stromal cells found within the diseased artery wall may promote the preferential recruitment of monocytes and this is achieved by establishing a cascade of interactions between SMCs, ECs, platelets, and monocytes.

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-alpha, 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-D(2) (PGD(2)) 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 PGD(3). This alternative series eicosanoid inhibited the migration of neutrophils across endothelial cells by antagonising the PGD(2) 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. PGD(2) 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.