G.E. Rainger

Prolonged E-selectin induction by monocytes potentiates the adhesion of flowing neutrophils to cultured endothelial cells

We investigated the hypothesis that the infiltration of monocytes into inflamed tissue or damaged vessels would induce a secondary accumulation of neutrophils. Confluent human umbilical vein endothelial cells (HUVEC) and blood monocytes (0.5 or 0.05 monocytes/endothelial cell) were co‐incubated for 4 or 24 h. The adhesion of neutrophils flowing over HUVEC was then analysed by video microscopy. Co‐incubation caused up to a 40‐fold increase in neutrophil adhesion, dependent upon monocyte/HUVEC ratio and duration of incubation. At the lower monocyte/HUVEC ratio, rolling adhesion alone was induced after 4 h co‐incubation; however, the full repertoire of rolling, immobilization and migration of neutrophils was observed at all other combinations of co‐culture ratio and exposure time. After maximal stimulation by monocytes, antibody blockade of the neutrophil integrin CD18 inhibited neutrophil arrest and migration and revealed underlying rolling adhesion. Rolling was supported by endothelial E‐selectin as demonstrated by the almost total abolition of adhesion by a blocking antibody. In a direct comparison, monocytes, tumour necrosis factor α (TNF‐α) and interleukin‐1β (IL‐1β) were assessed for their ability to induce endothelial expression of E‐selectin. E selectin was significantly increased by all agents at 4 h, but monocytes alone were able to maintain high levels of E‐selectin expression for 24 h. We conclude that monocytes can induce prolonged neutrophil adhesion and migration by activating endothelial cells and causing expression of E‐selectin.

Direct observations of the kinetics of migrating T-cells suggest active retention by endothelial cells with continual bidirectional migration.

The kinetics and regulatory mechanisms of T cell migration through the endothelium have not been fully defined. In experimental, filter‐based assays in vitro, transmigration of lymphocytes takes hours, compared with minutes, in vivo. We cultured endothelial cell (EC) monolayers on filters, solid substrates, or collagen gels and treated them with TNF‐α, IFN‐γ, or both prior to analysis of lymphocyte migration in the presence or absence of flow. PBL, CD4+ cells, or CD8+ cells took many hours to migrate through EC‐filter constructs for all cytokine treatments. However, direct microscopic observations of EC filters, which had been mounted in a flow chamber, showed that PBL crossed the endothelial monolayer in minutes and were highly motile in the subendothelial space. Migration through EC was also observed on clear plastic, with or without flow. After a brief settling without flow, PBL and isolated CD3+ or CD4+ cells crossed EC in minutes, but the numbers of migrated cells varied little with time. Close observation revealed that lymphocytes migrated back and forth continuously across endothelium. Under flow, migration kinetics and the proportions migrating back and forth were altered little. On collagen gels, PBL again crossed EC in minutes and migrated back and forth but showed little penetration of the gel over hours. In contrast, neutrophils migrated efficiently through EC and into gels. These observations suggest a novel model for lymphoid migration in which EC support migration but retain lymphocytes (as opposed to neutrophils), and additional signal(s) are required for onward migration.

Prostaglandin D 2 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 PGD2 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 PGD2 to nonmigrated cells. Migration across BVEC or exogenous PGD2 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 PGD2.

Integrin-substrate interactions underlying shear-induced inhibition of the inflammatory response of endothelial cells

Conditioning of endothelial cells by shear stress suppresses their response to inflammatory cytokines. We questioned whether signalling through different integrin-matrix interactions, previously associated with the pathogenic effects of disturbed flow, supported the anti-inflammatory action of steady shear. Primary human endothelial cells were cultured on different substrates and exposed to shear stress (2.0Pa) for varying periods before stimulation with tumour necrosis factor-α (TNF). Shear-conditioning inhibited cytokine-induced recruitment of flowing neutrophils. However, the effect was similar for culture on collagen, laminin or fibronectin, even when seeding was reduced to 2 hours, and shear to 3 hours before TNF treatment (to minimise deposition of endothelial matrix). Nevertheless, in short- or longer-term cultures, reduction in expression of β(1)-integrin (but not β(3)-integrin) using siRNA essentially ablated the effect of shear-conditioning on neutrophil recruitment. Studies of focal adhesion kinase (FAK) phosphorylation, siRNA against FAK and a FAK-inhibitor (PF573228) indicated that FAK activity was an essential component downstream of β(1)-integrin. In addition, MAP-kinase p38 was phosphorylated downstream of FAK and also required for functional modification. Mechanotransduction through β(1)-integrins, FAK and p38 is required for anti-inflammatory effects of steady shear stress. Separation of the pathways which underlie pathological versus protective responses of different patterns of flow is required to enable therapeutic modification or mimicry, respectively.

Origin‐Specific Adhesive Interactions of Mesenchymal Stem Cells with Platelets Influence Their Behavior After Infusion

We investigated the adhesive behavior of mesenchymal stem cells (MSC) in blood, which might influence their fate when infused as therapy. Isolated human bone marrow MSC (BMMSC) or umbilical cord MSC (UCMSC) adhered efficiently from flow to the matrix proteins, collagen, or fibronectin, but did not adhere to endothelial selectins. However, when suspended in blood, BMMSC no longer adhered to collagen, while UCMSC adhered along with many aggregated platelets. Neither MSC adhered to fibronectin from flowing blood, although the fibronectin surface did become coated with a platelet monolayer. UCMSC induced platelet aggregation in platelet rich plasma, and caused a marked drop in platelet count when mixed with whole human or mouse blood in vitro, or when infused into mice. In contrast, BMMSC did not activate platelets or induce changes in platelet count. Interestingly, isolated UCMSC and BMMSC both adhered to predeposited platelets. The differences in behavior in blood were attributable to expression of podoplanin (an activating ligand for the platelet receptor CLEC‐2), which was detected on UCMSC, but not BMMSC. Thus, platelets were activated when bound to UCMSC, but not BMMSC. Platelet aggregation by UCMSC was inhibited by recombinant soluble CLEC‐2, and UCMSC did not cause a reduction in platelet count when mixed with blood from mice deficient in CLEC‐2. We predict that both MSC would carry platelets in the blood, but their interaction with vascular endothelium would depend on podoplanin‐induced activation of the bound platelets. Such interactions with platelets might target MSC to damaged tissue, but could also be thrombotic. Stem Cells 2018;36:1062–1074

57 Mesenchymal stem cells inhibit recruitment of flowing neutrophils and lymphocytes by endothelial cells: roles of interleukin-6 and transforming growth factor-

Mesenchymal stem cells (MSC) have been suggested to have protective effects through modulation of immune responses. We examined the hypothesis that MSC can modify the ability of endothelial cells (EC) to recruit neutrophils and lymphocytes. Using flow-based assays, leukocytes were perfused over HUVEC that had been cultured with MSC, either on opposite sides of porous filers or in direct contact for 24 h, and then stimulated with tumour necrosis factor-α (TNF-α) for 4 h or TNF-α and interferon-γ for 24 h. Cytokine-stimulated EC supported neutrophil and lymphocyte recruitment, and a combination of rolling, firm adhesion and transmigration was observed. EC cultured with MSC supported significantly lower total adhesion of both types of leukocytes, and the proportion of adherent leukocytes that migrated was reduced compared to EC monocultures. The effects were dependent on the number of MSC added, but were evident for ratios of MSC to EC <<0.1. Interestingly, antibody neutralisation of interleukin-6 (IL-6) or transforming growth factor (TGF)-β during co-culture largely abolished the inhibitory effects of MSC on recruitment of both types of leukocyte. These antibodies did not modify recruitment to cytokine-treated EC cultured alone. We conclude that MSC can adhere to and integrate with EC monolayers, and then inhibit the inflammatory recruitment of leukocytes. The inhibitory effects may be due to linked production and effects of IL-6 and TGF-ï□¢ in the co-culture milieu.

T-Cell Trafficking

Immune cell development and function occur in specialized immunological tissues, the function of which requires active cell migration and interactions between hematopoietic cells and underlying networks of stromal cells. These cells provide a scaffold on which immune cell migrate, provide microenvironments for efficient antigen presentation, and provide signals required for immune cell recruitment and survival. Technical advances in imaging technologies including multiphoton microscopy and 3D tissue reconstructions are being combined with computational approaches to provide new insights into the process of cell migration and function in immunological tissues.

187 THE PEPTIDE INHIBITOR OF TRANS-ENDOTHELIAL MIGRATION, PEPITEM, A NOVEL IMMUNE REGUALTORY AGENT, CONTROLS T-CELL TRAFFICKING DURING INFLAMMATION, A TONIC INHIBITORY PATHWAY THAT IS LOST IN CHRONIC DISEASE

T-cells are recruited from the blood into extra-vascular tissues during acute inflammation. However, in chronic inflammatory diseases, including atherosclerosis, an inappropriate accumulation of T-cells in the diseased tissue contributes to pathogenesis. Very little is known about the mechanisms by which T-cell trafficking is regulated during inflammation, and it is thus difficult to target this aspect of pathology for the development of new anti-atherogenic therapies. Here we describe a unique immune regulatory peptide that imposes a tonic inhibition of T-cell trafficking during inflammation. PEPtide Inhibitor of Trans-Endothelial Migration (PEPITEM) introduces a new paradigm into the pathways that regulate the inflammatory response. We propose that loss of this regulatory pathway makes the immune system ‘leaky’, allowing inappropriate access of T-cells to vulnerable tissues in chronic diseases. Lymphocyte trafficking was assessed in vitro using videomicroscopy on TNF-α/IFN-γ activated endothelial cells (EC) and lymphocytes isolated from healthy donors or patients with chronic inflammatory disease. In vivo, lymphocyte recruitment was assessed in a model of zymosan-driven peritoneal inflammation. PEPITEM was identified using mass spectrometry. Our studies began with an interest in adiponectin, an anti-inflammatory adipose tissue-derived cytokine. Using an in vitro migration assay, we observed that the migration of human lymphocytes was dose-dependently blocked by adiponectin. Adiponectin achieves its effects on T-cell migration by the induction of a novel mediator released from B-cells. Thus, the effect of adiponectin was lost when B cells are absent, but could be regained by the addition of supernatants from adiponectin stimulated B-cells. Interestingly, the B-cell derived product did not act directly on T-cells; rather, it stimulated EC to release the lipid mediator sphingosine-1-phosphate, which in turn inhibited the migration of T-cells. We used mass spectrometry to isolate a B-cell derived peptide, corresponding uniquely in the human genome to a proteolytic excision product of the 14.3.3ζδ protein. Synthetic PEPITEM could also effectively inhibit T-cell migration. In zymosan-induced peritonitis in the mouse, T-cell recruitment was significantly increased in a strain lacking B cells when compared to wild-type animals. This excess of T-cell recruitment was ameliorated by treatment with PEPITEM. Lymphocytes isolated from patients with chronic inflammatory disease (type-1-diabetes) were released from the inhibitory effects of adiponectin, but this regulatory pathway could be re-established by the addition of exogenous PEPITEM. We believe that PEPITEM and its associated pathway may have therapeutic efficacy in a number of disease scenarios including atherosclerosis.