One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes

Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fy(a)/Fy(b) blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy protein expressed in K562 cells has three N-linked oligosaccharide chains, which are mainly of complex-type. Here we report a one-step purification method of Duffy protein from human erythrocytes. DARC was extracted from erythrocyte membranes in the presence of 1% n-dodecyl-β-D-maltoside (DDM) and 0.05% cholesteryl hemisuccinate (CHS) and purified by affinity chromatography using immobilized anti-Fy6 2C3 mouse monoclonal antibody. Duffy glycoprotein was eluted from the column with synthetic DFEDVWN peptide containing epitope for 2C3 monoclonal antibody. In this single-step immunoaffinity purification method we obtained highly purified DARC, which migrates in SDS-polyacrylamide gel as a major diffuse band corresponding to a molecular mass of 40-47?kDa. In ELISA purified Duffy glycoprotein binds anti-Duffy antibodies recognizing epitopes located on distinct regions of the molecule. Results of circular dichroism measurement indicate that purified DARC has a high content of α-helical secondary structure typical for chemokine receptors. Analysis of DARC glycans performed by means of lectin blotting and glycosidase digestion suggests that native Duffy N-glycans are mostly triantennary complex-type, terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core.     

P-selectin Cross-links PSGL-1 and Enhances Neutrophil Adhesion to Fibrinogen and ICAM-1 in a Src Kinase-Dependent,but GPCR-Independent Mechanism

Endothelial and platelet P-selectin (CD62P) and leukocyte integrin αMβ2 (CD11bCD18, Mac-1) are cell adhesion molecules essential for host defense and innate immunity. Upon inflammatory challenges, P-selectin binds to PSGL-1 (P-selectin glycoprotein ligand-1, CD162) to mediate neutrophil rolling, during which integrins become activated by extracellular stimuli for their firm adhesion in a G-protein coupled receptor (GPCR)-dependent mechanism. Here we show that cross-linking of PSGL-1 by dimeric or multimeric forms of platelet P-selectin, P-selectin receptor-globulin, anti-PSGL-1 mAb and its F(ab’)2 induced adhesion of human neutrophils to fibrinogen (Fg) and intercellular cell adhesion molecule-1 (ICAM-1, CD54) and triggered a moderate clustering of αMβ2, but monomeric forms of soluble P-selectin and anti-PSGL-1 Fab did not. Interestingly, P-selectin did not induce a detectable interleukine-8 (IL-8) secretion (<0.1 ng/ml) in 30 minutes, whereas a high concentration of IL-8 (>50 ng/ml) was required to increase neutrophil adhesion to Fg. P-selectin-induced neutrophil adhesion was significantly inhibited by PP2 (a Src kinase inhibitor), but not by Pertussis toxin (PTX; a GPCR inhibitor). Activated platelets also increased neutrophil binding to fibrinogen and triggered tyrosine phosphorylation of cellular proteins. Our results indicate that P-selectin-induced integrin activation (Src kinase-dependent) is distinct from that elicited by cytokines, chemokines, chemoattractants (GPCR-dependent), suggesting that these two signal transduction pathways may cooperate for maximal activation of leukocyte integrins.     

Duffy antigen receptor for chemokines mediates chemokine endocytosis through a macropinocytosis-like process in endothelial cells

Background

The Duffy antigen receptor for chemokines (DARC) shows high affinity binding to multiple inflammatory CC and CXC chemokines and is expressed by erythrocytes and endothelial cells. Recent evidence suggests that endothelial DARC facilitates chemokine transcytosis to promote neutrophil recruitment. However, the mechanism of chemokine endocytosis by DARC remains unclear.

Methodology/Principal Findings

We investigated the role of several endocytic pathways in DARC-mediated ligand internalization. Here we report that, although DARC co-localizes with caveolin-1 in endothelial cells, caveolin-1 is dispensable for DARC-mediated ¹²⁵I-CXCL1 endocytosis as knockdown of caveolin-1 failed to inhibit ligand internalization. ¹²⁵I-CXCL1 endocytosis by DARC was also independent of clathrin and flotillin-1 but required cholesterol and was, in part, inhibited by silencing Dynamin II expression. ¹²⁵I-CXCL1 endocytosis was inhibited by amiloride, cytochalasin D, and the PKC inhibitor Gö6976 whereas Platelet Derived Growth Factor (PDGF) enhanced ligand internalization through DARC. The majority of DARC-ligand interactions occurred on the endothelial surface, with DARC identified along plasma membrane extensions with the appearance of ruffles, supporting the concept that DARC provides a high affinity scaffolding function for surface retention of chemokines on endothelial cells.

Conclusions/Significance

These results show DARC-mediated chemokine endocytosis occurs through a macropinocytosis-like process in endothelial cells and caveolin-1 is dispensable for CXCL1 internalization.     

Duffy antigen facilitates movement of chemokine across the endothelium in vitro and promotes neutrophil transmigration in vitro and in vivo

The Duffy Ag expressed on RBCs, capillaries, and postcapillary venular endothelial cells binds selective CXC and CC chemokines with high affinity. Cells transfected with the Duffy Ag internalize but do not degrade chemokine ligand. It has been proposed that Duffy Ag transports chemokines across the endothelium. We hypothesized that Duffy Ag participates in the movement of chemokines across the endothelium and, by doing so, modifies neutrophil transmigration. We found that the Duffy Ag transfected into human endothelial cells facilitates movement of the radiolabeled CXC chemokine, growth related oncogene-alpha/CXC chemokine ligand 1 (GRO-alpha/CXCL1), across an endothelial monolayer. In addition, neutrophil migration toward GRO-alpha/CXCL1 and IL-8 (IL-8/CXCL8) was enhanced across an endothelial monolayer expressing the Duffy Ag. Furthermore, GRO-alpha/CXCL1 stimulation of endothelial cells expressing the Duffy Ag did not affect gene expression by oligonucleotide microarray analysis. These in vitro observations are supported by the finding that IL-8/CXCL8-driven neutrophil recruitment into the lungs was markedly attenuated in transgenic mice lacking the Duffy Ag. We conclude that Duffy Ag has a role in enhancing leukocyte recruitment to sites of inflammation by facilitating movement of chemokines across the endothelium.     

RGS10 restricts upregulation by chemokines of T cell adhesion mediated by α4β1 and αLβ2 integrins

Chemokines rapidly and transiently upregulate α4β1 and αLβ2 integrin-mediated adhesion during T lymphocyte extravasation by activating Gα-dependent inside-out signaling. To limit and terminate Gα-mediated signaling, cells can use several mechanisms, including the action of regulator of G protein signaling (RGS) proteins, which accelerate the GTPase activity of Gα subunits. Using human T cells silenced for or overexpressing RGS10, we show in this article that RGS10 functions as an inhibitor of Gα(i)-dependent, chemokine-upregulated T cell adhesion mediated by α4β1 and αLβ2. Shear stress-dependent detachment and cell spreading analyses revealed that RGS10 action mainly targets the adhesion strengthening and spreading phases of α4β1-mediated cell attachment. Associated with these observations, chemokine-stimulated Vav1-Rac1 activation was longer sustained and of higher intensity in RGS10-silenced T cells, or inhibited in cells overexpressing RGS10. Of importance, expression of constitutively activated Rac1 forms in cells overexpressing RGS10 led to the rescue of CXCL12-stimulated adhesion to VCAM-1 to levels similar to those in control transfectants. Instead, adhesion under flow conditions, soluble binding experiment, flow cytometry, and biochemical analyses revealed that the earlier chemokine-triggered integrin activation step was mostly independent of RGS10 actions. The data strongly suggest that RGS10 opposes activation by chemokines of the Vav1-Rac1 pathway in T cells, leading to repression of adhesion strengthening mediated by α4β1. In addition to control chemokine-upregulated T cell attachment, RGS10 also limited adhesion-independent cell chemotaxis and activation of cdc42. These results identify RGS10 as a key molecule that contributes to the termination of Gα-dependent signaling during chemokine-activated α4β1- and αLβ2-dependent T cell adhesion.     

Low molecular weight hyaluronan mediated CD44 dependent induction of IL-6 and chemokines in human dermal fibroblasts potentiates innate immune response

Complex regulation of the wound healing process involves multiple interactions among stromal tissue cells, inflammatory cells, and the extracellular matrix. Low molecular weight hyaluronan (LMW HA) derived from the degradation of high molecular weight hyaluronan (HMW HA) is suggested to activate cells involved in wound healing through interaction with HA receptors. In particular, receptor CD44 is suggested to mediate cell response to HA of different MW, being the main cell surface HA receptor in stromal tissue and immune cells. However, the response of dermal fibroblasts, the key players in granulation tissue formation within the wound healing process, to LMW HA and their importance for the activation of immune cells is unclear. In this study we show that LMW HA (4.3 kDa) induced pro-inflammatory cytokine IL-6 and chemokines IL-8, CXCL1, CXCL2, CXCL6 and CCL8 gene expression in normal human dermal fibroblasts (NHDF) that was further confirmed by increased levels of IL-6 and IL-8 in cell culture supernatants. Conversely, NHDF treated by HMW HA revealed a tendency to decrease the gene expression of these cytokine and chemokines when compared to untreated control. The blockage of CD44 expression by siRNA resulted in the attenuation of IL-6 and chemokines expression in LMW HA treated NHDF suggesting the involvement of CD44 in LMW HA mediated NHDF activation. The importance of pro-inflammatory mediators produced by LMW HA triggered NHDF was evaluated by significant activation of blood leukocytes exhibited as increased production of IL-6 and TNF-α. Conclusively, we demonstrated a pro-inflammatory response of dermal fibroblasts to LMW HA that was transferred to leukocytes indicating the significance of LMW HA in the inflammatory process development during the wound healing process.     

Signal-dependent slow leukocyte rolling does not require cytoskeletal anchorage of P-selectin glycoprotein ligand-1 (PSGL-1) or integrin αLβ2

In inflamed venules, neutrophils roll on P- or E-selectin, engage P-selectin glycoprotein ligand-1 (PSGL-1), and signal extension of integrin α(L)β(2) in a low affinity state to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Cytoskeleton-dependent receptor clustering often triggers signaling, and it has been hypothesized that the cytoplasmic domain links PSGL-1 to the cytoskeleton. Chemokines cause rolling neutrophils to fully activate α(L)β(2), leading to arrest on ICAM-1. Cytoskeletal anchorage of α(L)β(2) has been linked to chemokine-triggered extension and force-regulated conversion to the high affinity state. We asked whether PSGL-1 must interact with the cytoskeleton to initiate signaling and whether α(L)β(2) must interact with the cytoskeleton to extend. Fluorescence recovery after photobleaching of transfected cells documented cytoskeletal restraint of PSGL-1. The lateral mobility of PSGL-1 similarly increased by depolymerizing actin filaments with latrunculin B or by mutating the cytoplasmic tail to impair binding to the cytoskeleton. Converting dimeric PSGL-1 to a monomer by replacing its transmembrane domain did not alter its mobility. By transducing retroviruses expressing WT or mutant PSGL-1 into bone marrow-derived macrophages from PSGL-1-deficient mice, we show that PSGL-1 required neither dimerization nor cytoskeletal anchorage to signal β(2) integrin-dependent slow rolling on P-selectin and ICAM-1. Depolymerizing actin filaments or decreasing actomyosin tension in neutrophils did not impair PSGL-1- or chemokine-mediated integrin extension. Unlike chemokines, PSGL-1 did not signal cytoskeleton-dependent swing out of the β(2)-hybrid domain associated with the high affinity state. The cytoskeletal independence of PSGL-1-initiated, α(L)β(2)-mediated slow rolling differs markedly from the cytoskeletal dependence of chemokine-initiated, α(L)β(2)-mediated arrest.     

The Role of TLR2 and 4-Mediated Inflammatory Pathways in Endothelial Cells Exposed to High Glucose

Postprandial hyperglycemia induces inflammation and endothelial dysfunction resulting in vascular complications in patients with diabetes. Toll-like receptors (TLRs) are central to the regulation of inflammatory responses through activation of nuclear factor-kappa B (NF-ĸB). This study examined the role of TLR2 and 4 in regulating inflammation and endothelial dysfunction when exposed to fluctuating glucose concentrations. HMEC-1 cells (a human microvascular endothelial cell line) were exposed to control (5 mM), 30 mM (high), fluctuating (5/30 mM) and 11.2 mM glucose (approximate glycaemic criteria for the diagnosis of diabetes mellitus) for 72 h. Cells were assessed for TLR2, 4, high mobility group box -1 (HMGB1), NF-ĸB, monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Fluctuating glucose concentrations maximally upregulated TLR4 but not TLR2 expression with increased NF-ĸB activation, IL-8 and ICAM-1 expression. HMGB1 was increased in the supernatants of cells exposed to 30 mM and 11.2 mM glucose compared to control. The addition of recombinant HMGB1 induced NF-ĸB activation and synthesis of proinflammatory cytokines and chemokines, which were prevented by TLR2 or 4 signalling inhibition. An additive effect when both TLR2 and 4 signalling pathways were inhibited was observed. However, only inhibition of TLR4 signalling suppressed the synthesis of MCP-1, IL-8 and ICAM-1. In vivo, streptozotocin-induced diabetic mice exhibited an increase in glomerular ICAM-1 which was not evident in TLR2^-/- or TLR4^-/- diabetic mice. Collectively, our results suggest that targeting the signalling pathway of TLR2 and 4 may be of therapeutic benefit in attenuating vascular inflammation in diabetic microangiopathy.     

The immunomodulation of endotoxin-induced acute lung injury by hesperidin in vivo and in vitro

To investigate the modulation of lung local immune responses of hesperidin (HES) on the acute lung inflammation induced by LPS in vivo. Mice were challenged with intratracheal lipopolysaccharide (100 μg) 30 min before with treatment hesperidin (200 mg/kg oral administration) or vehicle. After 4 and 24 h, bronchoalveolar lavage fluid was obtained to measure proinflammatory (TNF-α, IL-1β, IL-6), anti-inflammatory (IL-10, IL-4, IL-12) cytokines, chemokines (KC, MCP-1 and MIP-2), total cell counts, nitric oxide production, and proteins. Lung histology was performed in inflated-fixed lungs. Hesperidin downregulate the LPS-induced expression of TNF-α, IL-1β, IL-6, KC, MIP-2, MCP-1, and IL-12. It also enhanced the production of IL-4, IL-10. Total leukocyte counts; nitric oxide production, iNOS expression, and proteins were significantly decreased by hesperidin. In vitro, HES suppressed the expression of IL-8 on A549 cells and THP-1 cells, the expression of TNF-α, IL-1β, and IL-6 on THP-1 cells, the expression of ICAM-1 and VCAM-1 on A549 cells which effect cell adhesion function. The suppression of those molecules is controlled by NF-κB and AP-1, which are activated by IκB and MAPK pathways. HES inhibits those pathways, thereby suppressing the expression of IL-8, TNFα, IL-1β, IL-6, IL-12, ICAM-1 and VCAM-1. This study indicates that HES had a markedly immunomodulatory effect in a clinically relevant model of ARDS. Nevertheless, further investigations are required to determine the potential clinical usefulness of HES in the adjunctive therapy of ARDS.     

Contribution of Duffy antigen to chemokine function

In addition to classical G protein-coupled receptors (GPCRs), a group of alternative, “silent” chemokine receptors has recently been identified. These serpentine molecules are not coupled to G proteins and subsequent signaling cascades, but can efficiently internalize their cognate chemokine ligands, thus act as “interceptors” (internalizing receptors). Here we discuss a mechanism by which a member of this family, Duffy antigen (DARC), contributes to chemokine-induced leukocyte emigration. Cumulative experimental evidence suggests that DARC on venular endothelium mediates chemokine internalization at the abluminal surface followed by transcytosis and transfer of the chemokine cargo onto the luminal surface. DARC is also expressed on the erythrocyte surface of DARC positive individuals. Erythrocyte DARC binds plasma chemokines which results, on one hand, in impediment of the chemokines loss from the circulation and, on the other hand, in neutralization of chemokines in the blood. This leads to leukocyte protection from inadvertent “desensitization” and enhancement of leukocyte recruitment.     

Divergent effects of nitric oxide on airway epithelial cell activation

Nitric oxide (NO*) is a gaseous mediator synthesized by nitric oxide synthases. NO* is involved in the modulation of inflammation, but its role in airway inflammation remains controversial. We investigated the role of NO* in the synthesis of the chemokines interleukin-8 and monocyte chemotactic protein-1, and of intercellular adhesion molecule-1 by human airway epithelial cells. normal human bronchial epithelial cells and the bronchial epithelial cell line BEAS-2B were used. interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) secretion and intercellular adhesion molecule-1 (ICAM-1) expression were measured by ELISA. mRNA was assessed by semiquantitative RTI-PCR. Interleukin-8 secretion was significantly reduced after 24h incubation with the NO* donor, sodium nitroprusside. The effect was dose-dependent. Similar results were obtained with S-nitroso-N-D,L-penicillamine and S-nitroso-L-glutathione. Inhibition of endogenous NO* with the nitric oxide synthase inhibitor N-nitro-L-arginine-methyl-ester caused an increase in IL-8 secretion by lipopolysaccharide- and cytokine-stimulated BEAS-2B cells. Sodium nitroprusside also caused a reduction in monocyte chemotactic protein-1 secretion by both cell types. In contrast, intercellular adhesion molecule-1 expression was upregulated by sodium nitroprusside. RTI-PCR results indicate that the modulation of protein levels was paralleled by modification in mRNA levels. NO* has divergent effects on the synthesis of different inflammatory mediators in human bronchial epithelial cells.     

Sulphated tyrosines mediate association of chemokines and Plasmodium vivax Duffy binding protein with the Duffy antigen/receptor for chemokines (DARC)

Plasmodium vivax is one of four Plasmodium species that cause human malaria. P. vivax and a related simian malaria parasite, Plasmodium knowlesi, invade erythrocytes by binding the Duffy antigen/receptor for chemokines (DARC) through their respective Duffy binding proteins. Here we show that tyrosines 30 and 41 of DARC are modified by addition of sulphate groups, and that the sulphated tyrosine 41 is essential for association of the Duffy binding proteins of P. vivax (PvDBP) and P. knowlesi (PkDaBP) with DARC-expressing cells. These sulphated tyrosines also participate in the association of DARC with each of its four known chemokine ligands. Alteration of tyrosine 41 to phenylalanine interferes with MCP-1, RANTES and MGSA association with DARC, but not with that of IL8. In contrast, alteration of tyrosine 30 to phenylalanine interferes with the association of IL8 with DARC. A soluble sulphated amino-terminal domain of DARC, but not one modified to phenylalanine at residue 41, can be used to block the association of PvDBP and PkDaBP with red blood cells, with an IC50 of approximately 5 nM. These data are consistent with a role for tyrosine sulphation in the association of many or most chemokines with their receptors, and identify a key molecular determinant of erythrocyte invasion by P. vivax.     

αvβ5/β6 integrin suppression leads to a stimulation of α2β1 dependent cell migration resistant to PI3K/Akt inhibition

Crosstalk between integrins is involved in the regulation of various cell functions including cell migration. Here we identify the interplay between the integrins αvβ5/β6 and α2β1 during cell migration toward type I collagen. Human colon cancer cell lines HT29-D4 and SW480 were used as cell models. To improve our understanding of the consequences of αvβ5/β6 function on α2β1, we decreased the expression of αv integrins by either siRNA or lysosomal targeting strategies or inhibited their function using, as antagonists, blocking antibodies or disintegrins. In all cases, we observed a greatly enhanced α2β1 integrin-dependent cell migration associated with focal adhesion rearrangements and increased outside-in signaling as demonstrated by elevated phosphorylation of focal adhesion kinase and MAPKinase (ERK1 and ERK2). The αvβ5/β6-dependent limitation of α2β1 function could be overridden by TS2/16, an activating anti-β1 antibody. Interestingly, compared to control cells, the pharmacological inhibition of PI3Kinase or the siRNA-mediated knockdown of AKT had little effect on the high α2β1-mediated cell migration observed in the absence of αv integrins or following activation of α2β1 integrins by the TS2/16. These results suggest that integrins αvβ5/β6 repress α2β1 possibly by interfering with their activation process and thereby modify the cell signaling regulation of α2β1-mediated migration.     

In vitro inhibitory effect of IL-8 and other chemoattractants on neutrophil-endothelial adhesive interactions.

We have previously reported that cytokine- or LPS-activated human umbilical vein endothelial cell (HUVEC) monolayers secrete IL-8 that can act as a neutrophil-selective adhesion inhibitor. In our study we investigated the mechanisms involved in the leukocyte adhesion inhibitory action of IL-8. The leukocyte adhesion inhibitory effect appears to be mediated by the action of IL-8 on the neutrophil, does not involve down-regulation of relevant endothelial adhesion molecules such as endothelial-leukocyte adhesion molecule-1 or intercellular adhesion molecule-1, and is quantitatively similar in different endothelial activation states that are predominantly endothelial-leukocyte adhesion molecule-1 dependent or intercellular adhesion molecule-1 dependent. In addition to inhibiting the attachment of freshly isolated peripheral blood neutrophils to cytokine-activated HUVEC monolayers, IL-8 also promoted a rapid detachment of tightly adherent neutrophils from activated HUVEC, and abolished neutrophil transendothelial migration. Certain other chemoattractants, including FMLP and C5a, had similar inhibitory actions, indicating IL-8 was not unique in its ability to inhibit various neutrophil-endothelial interactions. In contrast, two other neutrophil agonists 1-0-alkyl-2-acetyl sn-glycero-3-phosphocholine and granulocyte-macrophage-CSF, which, like IL-8, are produced by activated HUVEC, as well as the leukocyte-derived chemoattractant leukotriene B4, exerted minimal inhibitory effects on adhesion. Regardless of their ability to modulate neutrophil-endothelial cell adhesion, all these agents induced altered leukocyte surface expression of functionally important adhesion molecules, including loss of L-selectin (leukocyte adhesion molecule-1, LECAM-1) and increase in CD11b/CD18. Thus, although the above agonists have been characterized primarily as chemoattractants, our findings demonstrate that these agents can exert a wide range of modulatory effects on neutrophil-endothelial adhesive interactions.     

RNA aptamer therapy for vaso-occlusion in sickle cell disease

Patients with sickle cell disease (SCD) often suffer painful vaso-occlusive episodes caused in part by the adhesion of sickle erythrocytes (SS-RBC) to the vascular endothelium. To investigate inhibition of SS-RBC adhesion as a possible treatment for vaso-occlusion, 2 adhesion molecules, α(v)β(3) and P-selectin, were targeted by high-affinity RNA aptamers. An in vitro flow chamber assay was used to test the antiadhesion activity of α(v)β(3) aptamer clone 17.16. Human SS-RBC were passed across a confluent monolayer of thrombin-stimulated human umbilical vein endothelial cells (HUVEC) at a constant rate. α(v)β(3) aptamer reduced SS-RBC adhesion to activated endothelial cells to the level seen with untreated HUVEC. An aptamer reactive with complement component 8 was used as a negative control and exerted no inhibition, confirming the specificity of α(v)β(3) aptamer (P=0.04). At 2?dyn/cm(2) shear stress, 30?nM α(v)β(3) aptamer showed maximal effect in decreasing SS-RBC adhesion to HUVEC. The antiadhesive activity of the P-selectin aptamer clone PF377 was also tested using HUVEC pretreated with IL-13 to upregulate expression of P-selectin as seen in activated endothelial cells. At 1?dyn/cm(2) shear stress, 60?nM of P-selectin aptamer had antiadhesion activity similar to heparin, a known inhibitor of SS-RBC adhesion to P-selectin. A negative control did not prevent adhesion (P=0.05). These data show the potential utility of aptamers to block endothelial adhesion molecules to prevent or treat vaso-occlusion in SCD.     

Different patterns of IL-1β secretion, adhesion molecule expression and apoptosis induction in human endothelial cells treated with 7α-, 7β-hydroxycholesterol, or 7-ketocholesterol

Among oxysterols oxidized at C7 (7α-, 7β-hydroxycholesterol, and 7-ketocholesterol), 7β-hydroxycholesterol and 7-ketocholesterol involved in the cytotoxicity of oxidized low density lipoproteins (LDL) are potent inducers of apoptosis. Here, we asked whether all oxysterols oxidized at C7 were able to trigger apoptosis, to stimulate interleukin (IL)-1β and/or tumor necrosis factor (TNF)-α secretion, and to enhance adhesion molecule expression (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin) on human umbilical venous endothelial cells (HUVECs). Only 7β-hydroxycholesterol and 7-ketocholesterol were potent inducers of apoptosis and of IL-1β secretion. TNF-α secretion was never detected. Depending on the oxysterol considered, various levels of ICAM-1, VCAM-1 and E-selectin expression were observed. So, oxysterols oxidized at C7 differently injure and activate HUVECs, and the α- or β-hydroxyl radical position plays a key role in apoptosis and IL-1β secretion.     

Effects of Antioxidant and Nitric Oxide on Chemokine Production in TNF-α-stimulated Human Dermal Microvascular Endothelial Cells

Chemokines have been implicated convincingly in the driving of leukocyte emigration in different inflammatory reactions. Multiple signaling mechanisms are reported to be involved in intracellular activation of chemokine expression in vascular endothelial cells by various stimuli. Nevertheless, redox-regulated mechanisms of chemokine expression in human dermal microvascular endothelial cells (HDMEC) remain unclear. This study examined the effects of pyrrolidine dithiocarbamate (PDTC, 0.1?mM) and spermine NONOate (Sper-NO, 1?mM) on the secretion and gene expression of chemokines, interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, regulated upon activation normal T cell expressed and secreted (RANTES), and eotaxin. This study also addresses PDTC and Sper-NO effects on activation of nuclear factor kappa B (NF-κB) induced by TNF-α (10?ng/ml). Treatment with TNF-α for 8?h significantly increased secretion of IL-8, MCP-1, and RANTES, but not of eotaxin, in cultured HDMEC. Up-regulation of these chemokines was suppressed significantly by pretreatment with PDTC or Sper-NO for 1?h, but not by 1?mM 8-bromo-cyclic GMP. The mRNA accumulation of IL-8, MCP-1, RANTES, and eotaxin, and activation of NF-κB were induced by TNF-α for 2?h; all were suppressed significantly by the above two pretreatments. These findings indicate that both secretion and mRNA accumulation of IL-8, MCP-1, and RANTES in HDMEC induced by TNF-α are inhibited significantly by pretreatment with PDTC or Sper-NO, possibly via blocking redox-regulated NF-κB activation. These results suggest that restoration of the redox balance using antioxidant agents or nitric oxide pathway modulators may offer new opportunities for therapeutic interventions in inflammatory skin diseases.