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PubMed2000

Protection from ischemia-reperfusion induced severe acute renal failure by blocking E-selectin.

OBJECTIVE: Despite progress in renal replacement therapy and critical care medicine, acute renal failure (ARF) still carries a very high mortality rate. Neutrophil infiltration has been recognized as a hallmark in postischemic renal injury. Neutrophil recruitment requires adhesion molecules including E-selectin, which mediates leukocyte rolling and adhesion. This study aims to identify the role of E-selectin in ischemia-reperfusion-induced severe ARF. DESIGN: Prospective, controlled, experimental study. SETTING: University animal research laboratory. SUBJECTS: C57BL/6 wild-type mice or C57BL/6 mice gene-deficient for E-selectin. INTERVENTIONS: Mice underwent 32-min bilateral renal ischemia or identical sham operations. After 4, 12, 24, or 48 hrs, kidneys were harvested and blood samples were taken. A separate group of wild-type mice received either antineutrophil serum or control serum 18 hrs before ischemia. Another group of wild-type mice was injected with function-blocking monoclonal E-selectin antibody or with control antibody 10 mins after reperfusion. Blood samples were taken 24 hrs later. MEASUREMENTS AND MAIN RESULTS: Blood creatinine and urea nitrogen concentrations, as well as renal myeloperoxidase activity indicating neutrophil infiltration, were measured. Reducing neutrophil counts by antineutrophil serum showed that in this model, organ failure strongly depends on neutrophil counts at time of ischemia. E-selectin deficient mice showed lower creatinine and blood urea nitrogen concentrations than wild-type mice at 24 and 48 hrs (a reduction of 60% to 80%). Kidneys of E-selectin deficient mice also revealed a lower myeloperoxidase activity maximum (75% reduction) at 24 hrs. Western blot analysis showed maximum E-selectin expression 24 hrs after ischemia-reperfusion. Immunostaining localized E-selectin to the endothelium of the peritubular capillary plexus. Compared with control antibody, postischemic injection of anti-E-selectin antibody gave lower creatinine concentrations at 24 hrs, similar to that seen in E-selectin deficient mice. CONCLUSIONS: In this model, blocking E-selectin even after onset of reperfusion protects from severe ARF, presumably by reducing postischemic neutrophil infiltration into the kidney. This suggests a new potential therapeutic perspective.

Acute Disease↗
PubMed2001

Soluble L-selectin at levels present in septic patients diminishes leukocyte-endothelial cell interactions in mice in vivo: a mechanism for decreased leukocyte delivery to remote sites in sepsis.

OBJECTIVE: Recent in vivo studies of both septic humans and animals demonstrate that leukocyte delivery is attenuated to sites remote from the primary infection. The mechanisms for this are not entirely clear. L-selectin is integral to rolling, the first step in leukocyte recruitment to an inflammatory site. L-selectin is shed from leukocytes in sepsis, resulting in increased levels of soluble L-selectin in plasma (2.33 microg/mL). This study investigates the effects of soluble L-selectin at levels found in sepsis on leukocyte trafficking in vivo. DESIGN: Prospective, controlled trial. SETTING: Surgical research laboratory in a university hospital. SUBJECTS: Swiss white male mice of 25-35 g. INTERVENTIONS: Mice were randomized to one of three study groups: soluble L-selectin 2.33, soluble L-selectin 8.0, or albumin. Intravital microscopy was performed on postcapillary venules of 20-40 microm in diameter in the cremaster muscle of mice. Leukocyte-endothelial cell interactions (rolling, adherence, and rolling velocity) were measured pre- and post- (1, 15, 30, and 45 mins) intravenous infusion of human recombinant soluble L-selectin (2.33 and 8.0 microg/mL) or human albumin (8.0 microg/mL). MEASUREMENTS AND MAIN RESULTS: The intravenous administration of soluble L-selectin to a systemic concentration of 2.33 microg/mL diminished rolling significantly. Soluble L-selectin at 8.0 microg/mL decreased rolling and increased rolling velocity to a greater degree. Injection of albumin did not alter leukocyte-endothelial cell interactions at any time point. No difference between groups in blood pressure, shear rate, or leukocyte counts was detected. CONCLUSIONS: Soluble L-selectin diminishes leukocyte rolling at levels present in sepsis (2.33 microg/mL). This effect is dose dependent, and could not be explained by differences in blood pressure, shear rate, or leukocyte counts. These findings identify increased soluble L-selectin levels as one of the mechanisms for decreased leukocyte delivery and exudation to remote sites in septic patients.

Analysis of Variance↗
PubMed2005

Plasma soluble adhesion molecules; intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and E-selectin levels in patients with isolated coronary artery ectasia.

UNLABELLED: Plasma soluble adhesion molecules, intercellular adhesion molecule-1 (ICAM)-1, vascular cell adhesion molecule-1 (VCAM-1) and E-selectin leves of patients with isolated coronary artery ectasia (CAE), patients with obstructive coronary artery disease without CAE and subjects with angiographically normal coronary arteries were evaluated. Patients with isolated CAE were detected to have significantly higher levels of plasma soluble ICAM-1, VCAM-1 and E-selectin in comparison with patients with obstructive coronary artery disease without CAE (ICAM, 673 +/- 153 versus 381 +/- 106, respectively, P < 0.001; VCAM-1, 2366 +/- 925 versus 1136 +/- 208, respectively, P < 0.001; E-selectin, 74 +/- 21 versus 61 +/- 18, respectively, P = 0.01) and subjects with normal coronary arteries (ICAM-1, 673 +/- 153 versus 303 +/- 131, respectively, P < 0.001; VCAM-1, 2366 +/- 925 versus 729 +/- 231, respectively, P < 0.001; E-selectin, 74 +/- 21 versus 49 +/- 9, respectively, P < 0.001), suggesting the presence of a more severe and extensive chronic inflammation in the coronary circulation in patients with isolated CAE. BACKGROUND: The common coexistence of coronary artery ectasia (CAE) with coronary artery disease (CAD) suggests that it may be a variant of CAD. However, it is not clear why some patients with obstructive CAD develop CAE whereas most do not. Inflammation has been reported to be a major contributing factor to both obstructive and aneurysmatic vascular disorders and therefore, in the present study, the plasma soluble adhesion molecules, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin levels in isolated CAE were investigated. METHODS: The study population consisted of three groups: the first consisted of 32 patients with isolated CAE without stenotic lesion; the second of 32 patients with obstructive CAD without CAE; and the third group of 30 control subjects with normal coronary arteries. Coronary diameters were measured as the maximum diameter of the ectasic segment by use of a computerized quantitative coronary angiography analysis system. According to the angiographic definition used in the Coronary Artery Surgery Study, a vessel is considered to be ectasic when its diameter is > or =1.5 times that of the adjacent normal segment in segmental ectasia. Plasma soluble ICAM-1, VCAM-1 and E-selectin levels were measured in all patients and control subjects using commercially available enzyme-linked immunosorbent assay kits. RESULTS: Patients with isolated CAE were found to have significantly higher levels of plasma soluble ICAM-1, VCAM-1, and E-selectin in comparison with patients with obstructive CAD without CAE (ICAM, 673 +/- 153 versus 381 +/- 106, respectively; P < 0.001; VCAM-1, 2366 +/- 925 versus 1136 +/- 208, respectively; P < 0.001; E-selectin, 74 +/- 21 versus 61+/-18, respectively; P = 0.01) and control subjects with normal coronary arteries (ICAM-1, 673 +/- 153 versus 303 +/- 131, respectively;, P < 0.001; VCAM-1, 2366 +/- 925 versus 729 +/- 231, respectively; P < 0.001; E-selectin, 74 +/- 21 versus 49 +/- 9, respectively; P < 0.001). In addition, we detected statistically significant positive correlation between the total length of ectasic segments and the levels of plasma soluble ICAM-1 (r = 0.625; P < 0.001), VCAM-1 (r = 0.548; P = 0.001) and E-selectin (r = 0.390; P = 0.027). Multivariate logistic regression analysis revealed a significant independent relation between isolated CAE and ICAM-1 [odds ratio (OR) = 1.023; 95% confidence interval (CI) = 1.0048-1.0414; P = 0.0129] and VCAM-1 (OR = 1.0057; 95% CI = 1.0007-1.0106; P = 0.0240). CONCLUSIONS: We have shown that patients with isolated CAE have raised levels of plasma soluble ICAM-1, VCAM-1 and E-selectin in comparison with patients with obstructive CAD without CAE and control subjects with normal coronary arteries, suggesting the presence of a more severe and extensive chronic inflammation in the coronary circulation in these patients.

Adult↗
PubMed2005

PSGL-1 derived from human neutrophils is a high-efficiency ligand for endothelium-expressed E-selectin under flow.

P-selectin glycoprotein ligand-1 (PSGL-1) has been proposed as an important tethering ligand for E-selectin and is expressed at a modest level on human leukocytes. Sialyl Lewis x (sLe(x))-like glycans bind to E-selectin and are expressed at a relatively high level on circulating leukocytes. It is unclear whether PSGL-1 has unique biochemical attributes that contribute to its role as an E-selectin ligand. To probe this issue, we conjugated microspheres with either sLe(x) or PSGL-1 purified from myeloid cells (neutrophils and HL-60) and compared their adhesion to endothelial expressed E-selectin under defined shear conditions. We found that both sLe(x) and PSGL-1 microspheres adhere to 4 h of IL-1beta-activated human umbilical vein endothelial cells predominantly through E-selectin. Analysis of the adhesion revealed that the rate of initial tethering of the PSGL-1 microspheres to E-selectin was significantly greater than the rate of initial tethering of the sLe(x) microspheres despite the fact that the sLe(x) microspheres tested had higher ligand densities than the PSGL-1 microspheres. We also found that pretreatment of the PSGL-1 or sLe(x) microspheres with HECA-452 had no significant effect on initial tethering to E-selectin. These results support the hypotheses that 1) PSGL-1 is a high-efficiency tethering ligand for E-selectin, 2) ligand biochemistry can significantly influence initial tethering to E-selectin, and 3) PSGL-1 tethering to E-selectin can occur via non-HECA-452 reactive epitopes.

Cell Adhesion↗
PubMed2006

Death receptor-3, a new E-Selectin counter-receptor that confers migration and survival advantages to colon carcinoma cells by triggering p38 and ERK MAPK activation.

E-selectin-mediated adhesion of colon cancer cells to endothelial cells is a key event in metastasis. However, the signaling mechanisms that confer metastatic advantages to cancer cells adhering to E-selectin are ill defined. By using affinity column chromatography and pull-down assays on purified membrane extracts of HT29 and LoVo cells coupled to mass spectrometry analysis, we obtained the first evidence indicating that E-selectin binds to death receptor-3 (DR3) expressed by the cancer cells. Thereafter, we accumulated several results, suggesting that DR3 is an E-selectin receptor on colon cancer cells and that its activation by E-selectin triggers the activation of p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) and confers migration and survival advantages. First, by Western blotting, we found that the E-selectin-binding protein, identified as DR3, is recognized by two anti-DR3 antibodies. Second, the neutralization of DR3 with an antibody and its knockdown by small interfering RNA decrease the adhesion of colon cancer cells to E-selectin and E-selectin-expressing human umbilical vein endothelial cells. Third, inhibiting DR3 and knocking down its expression impair transendothelial migration of HT29 cells and block the activation of p38 and ERK by E-selectin. Fourth, high molecular weight isoforms of DR3 are expressed in samples of primary human colon carcinoma but not in samples from normal colon tissue. Intriguingly, DR3 is a death receptor but its activation by E-selectin does not induce apoptosis in colon cancer cells, except when ERK is inhibited. Our findings identify novel signaling and functional roles of DR3 activated in response to E-selectin and highlight the potential link between DR3 and metastasis.

Apoptosis↗
PubMed2004

E-Selectin and restenosis after femoropopliteal angioplasty: prognostic impact of the Ser128Arg genotype and plasma levels.

Perivascular inflammation plays a key role in the development of restenosis after percutaneous transluminal angioplasty (PTA). The adherence of leucocytes to the activated endothelium, an essential feature in the restenotic process, is mediated by the cellular adhesion molecule E-Selectin. A DNA polymorphism in the regulator region of E-Selectin at codon 561 (Ser128Arg) is suggested to modulate the molecule's physiological effects. Therefore, we investigated the association between the E-Selectin Ser128Arg genotype, E-Selectin plasma levels and restenosis after femoropopliteal PTA. We prospectively studied 175 consecutive patients with peripheral artery disease and intermittent claudication (n=126) or critical limb ischemia (n=49) who underwent primary successful femoropopliteal balloon angioplasty. E-Selectin Ser128Arg genotype and base-line E-Selectin plasma levels were determined and patients were followed up for median 12 months (IQR 11 to 14, total range 6 to 24) for the occurrence of postangioplasty restenosis(>/=50%). E-Selectin plasma levels in homozygous Arg128Arg and heterozygous Ser128Arg patients were significantly higher compared to wildtype Ser128Ser patients (p=0.041). Patency rates for wildtype Ser128Ser, heterozygous Ser128Arg and homozygous Ser128Ser patients were 57%, 44% and 50% at 6 months, and 46%, 40% and 17%, at 12 months, respectively (Log Rank p=0.31). Patency rates for increasing tertiles of E-Selectin were 61%, 58% and 37% at 6 months, and 54%, 45% and 30% at 12 months, respectively (Log Rank p=0.020). Patients with an E-Selectin plasma level above 44.9 mg/dL (third tertile) had an 1.9-fold increased adjusted risk for restenosis (95% CI 1.09 to 3.30). E-Selectin plasma levels are modulated by the E-Selectin Ser128Arg genotype, and predict the risk for restenosis after PTA in patients with PAD. A direct association of the Ser128Arg polymorphism with late postangioplasty failure could not be demonstrated.

Aged↗
PubMed1995

Inhibition of nitric oxide biosynthesis promotes P-selectin expression in platelets. Role of protein kinase C.

Inhibition of NO synthesis promotes P-selectin expression on endothelial cells; however, the precise mechanism is unclear. Because No has been shown to inhibit protein kinase C (PKC) activity, we examined the hypothesis that the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) stimulates P-selectin expression on platelets via PKC activation. Ten-minute incubation with either phorbol 12-myristate 13-acetate (PMA), thrombin, or L-NAME significantly increased P-selectin expression on platelets (as assessed by flow-cytometric analysis) and PKC activity of platelet membranes. Increased P-selectin expression induced by either PMA, thrombin, or L-NAME was significantly attenuated by the selective PKC inhibitor UCN-01 (7-hydroxystaurosporine). Furthermore, L-NAME-induced P-selectin expression was significantly attenuated by either L-arginine, 8-bromo-cGMP, or sodium nitroprusside (SNP). Interestingly, L-NAME further potentiated P-selectin upregulation by thrombin. L-NAME, thrombin, and PMA also significantly increased polymorphonuclear leukocyte adherence to the coronary artery endothelium, an effect that was significantly attenuated by the anti-P-selectin monoclonal antibody PB1.3 or by UCN-01, L-arginine, 8-bromo-cGMP or SNP but not by D-arginine or he nonblocking anti-P-selectin monoclonal antibody NBP1.6. These results indicate that inhibition of NO synthesis induces rapid P-selectin expression, which appears to be at least partially mediated by PKC activation in platelets. Similar effects and mechanisms of L-NAME on P-selectin function were also observed in endothelial cells, another site of P-selectin expression.

Animals↗
PubMed1999

Stat6 activation is essential for interleukin-4 induction of P-selectin transcription in human umbilical vein endothelial cells.

Chronic upregulation of P-selectin expression on the surface of the endothelium has been observed in and likely contributes to a number of chronic inflammatory diseases, including atherosclerosis. Agonists of P-selectin expression fall into 2 categories: those that induce a very rapid, transient increase, lasting only hours, and those that induce prolonged upregulation lasting days. It is the latter group, which includes interleukin-4 (IL-4), that is likely to be a mediator of chronic P-selectin upregulation. The increase in P-selectin expression induced by IL-4 results from increased transcriptional activation of the P-selectin gene. The aim of this study was to deduce the postreceptor signaling pathway(s) giving rise to the prolonged increase in P-selectin expression induced by IL-4. We demonstrate the existence of 2 functional signal transducer and activator of transcription 6 (Stat6) binding sites on the P-selectin promoter and further demonstrate, by functional analysis of the P-selectin promoter, that binding of activated Stat6 to at least 1 site is essential for IL-4-induction of P-selectin transcription. Site 1 (nucleotide[nt] -142) bound Stat6 with a higher affinity than did site 2 (nt -229), and this difference was reflected functionally as constructs in which only site 1 was functional showed full IL-4 inducibility, whereas constructs in which only site 2 was functional showed only 40% of maximal IL-4 inducibility. IL-4 also induced prolonged activation of Stat6, which was contingent on the continuous presence of IL-4. The sustained activation of Stat6 induced by IL-4 is likely to be a key factor leading to the prolonged activation of the P-selectin promoter, thereby resulting in prolonged P-selectin upregulation.

Base Sequence↗
PubMed2000

A role for platelets and endothelial selectins in tumor necrosis factor-alpha-induced leukocyte recruitment in the brain microvasculature.

The mechanisms mediating leukocyte recruitment into the cerebral nervous system during inflammation are still poorly understood. The objective of this study was to investigate the leukocyte recruitment in the brain microcirculation by intravital microscopy. Superfusion of the brain with artificial cerebrospinal fluid did not induce leukocyte rolling or adhesion. However, intraperitoneal tumor necrosis factor-alpha (TNF-alpha) caused marked leukocyte rolling and adhesion in the brain microcirculation. Histology revealed that the recruitment was primarily of neutrophils. Both E- and P-selectin were required for TNF-alpha-induced leukocyte recruitment, as rolling was reduced after treatment with either anti-E- or anti-P-selectin antibody and eliminated in E- or P-selectin-deficient mice. A significant increase in brain P- and E-selectin expression was seen after TNF-alpha treatment, but both were an order of magnitude less than in any other tissue. We observed significant platelet paving of TNF-alpha-stimulated endothelium and found that anti-platelet antibody reduced leukocyte rolling and adhesion, as did acetylsalicylic acid (aspirin). However, depletion of platelets did not reduce cerebral P-selectin expression. Moreover, chimeric mice lacking P-selectin on endothelium but not platelets had significantly decreased P-selectin expression and reduced leukocyte recruitment in the brain. This suggests a role for endothelial P-selectin in cerebral leukocyte recruitment. In conclusion, TNF-alpha-induced neutrophil recruitment into the brain requires both endothelial E-selectin and P-selectin as well as platelets, but platelet P-selectin was not a major contributor to this process.

Animals↗
PubMed1998

Antagonism of selectin-dependent adhesion of human eosinophils and neutrophils by glycomimetics and oligosaccharide compounds.

Early in inflammation, adhesion occurs between leukocytes and endothelium when selectins bind to sialyl Lewis X (sLex) and related oligosaccharides. We tested novel compounds that mimic sLex for their ability to inhibit selectin-mediated adhesion of human eosinophils and neutrophils in vitro. Neutrophils and eosinophils were isolated by density gradient centrifugation, and eosinophils were further purified by immunomagnetic negative selection. Adhesion to unstimulated or interleukin-1beta-stimulated (5 ng/ml, 4-6 h) umbilical vein endothelial monolayers was tested under static or rotating conditions, where adhesion is primarily E- or L-selectin dependent, respectively. P-selectin-dependent adhesion was tested on immobilized platelets treated with or without phorbol myristate acetate (10(-7) M, 10 min). Stimulus-induced adhesion was always at least 4-fold higher than without stimulus, and selectin dependence was confirmed with specific blocking monoclonal antibodies. E-selectin-dependent adhesion of eosinophils and neutrophils was inhibited by compound GM2296 (the concentration producing 50% inhibition of adhesion [IC50] approximately 0.5-1 mM). E-selectin-dependent adhesion of neutrophils, but not eosinophils, was also inhibited by another compound, sLex with a lipid tail (30 +/- 6% inhibition at 3 mM), whereas compound GM1292 slightly inhibited adhesion of both (23 +/- 5 and 20 +/- 6% inhibition, respectively, at 1 mM). L-selectin-dependent adhesion was more effectively inhibited by GM2296 (IC50 approximately 0.2-0.5 mM), although P-selectin-dependent adhesion was also inhibited (IC50 approximately 1 mM). Inhibition was reversible without affecting viability, and no effect was seen with these compounds in assays testing neutrophil adhesion to immobilized intercellular adhesion molecule-1. Thus, compound GM2296, a carbon-fucosylated derivative of glycyrrhetinic acid, inhibits E-, L-, and P-selectin-dependent eosinophil and neutrophil adhesion. The ability of these and perhaps other related glycomimetic compounds to interfere with the function of more than one type of selectin makes them desirable candidates as anti-inflammatory agents.

Antibodies, Monoclonal↗
PubMed1997

A minimal role for selectins in the recruitment of leukocytes into the inflamed liver microvasculature.

A two-step paradigm for leukocyte recruitment has been established in a number of tissues including the mesentery, skin, and muscle, and necessitates an initial rolling step via the selectins before firm leukocyte adhesion via the integrins. In view of the many inflammatory diseases that involve the liver, we investigated the importance of rolling and the selectins in the hepatic microvasculature and compared the responses to that of the commonly used mesentery or cremaster microvasculature. We visualized the liver microvasculature using intravital microscopy and we determined that within the liver the majority of leukocytes adhere within the sinusoids (80%) in response to a chemotactic stimulus such as FMLP (20% in postsinusoidal venules) whereas leukocytes adhere exclusively within postcapillary venules in tissue like the mouse cremaster. In the sinusoids, the adhesive response to FMLP is not dependent upon selectins inasmuch as adhesion was not reduced in the sinusoidal vessels of P-selectin-deficient mice or E-selectin/P-selectin- deficient animals in the presence or absence of L-selectin antibody. No rolling or adhesion was detected in response to FMLP in the selectin-deficient cremaster microvasculature. Immunoneutralization of selectins with fucoidan in wildtype mice eliminated rolling and adhesion in the cremaster but failed to affect adhesion in the liver sinusoids in response to FMLP. More long-term leukocyte recruitment with lipopolysaccharide (4 h) was also impaired in the cremaster but not the liver microvasculature in selectin-deficient animals. Leukocyte adhesion in the sinusoids was reduced in P-selectin-deficient mice also lacking intercellular adhesion molecule-1 (ICAM-1). This study for the first time demonstrates that selectins are not an essential step for leukocyte recruitment into the inflamed liver microvasculature.

Animals↗
PubMed2002

Protective effects of inhibiting both blood and vascular selectins after stroke and reperfusion.

Early intervention after acute ischemic stroke is essential to minimize brain cell injury. Although reperfusion of the ischemic brain is the treatment of choice for acute stroke, reperfusion itself may cause additional injury. The inflammatory cascade, characterized in part by early leukocyte interaction with endothelium, may contribute to this additional injury to blood vessels and surrounding brain tissue, extending the area of infarction. The selectin family of adhesion molecules mediates the initial, rolling and tethering of leukocytes to endothelium. P-selectin is rapidly expressed on ischemic endothelium in the brain vasculature, and L-selectin is expressed on leukocytes. Blocking the selectin-mediated tethering step may limit the inflammatory component of reperfusion injury in the brain. Fucoidin (FCN), a competitive inhibitor of P- and L-selectin, has been reported to decrease leukocyte accumulation during reperfusion of other organs. The effect of both leukocyte and endothelial selectin inhibition after cerebral ischemia and reperfusion has not been previously examined. The purpose of this study was to determine the effects of selectin adhesion molecule blockade on cerebral infarction size and neurological function after middle cerebral artery occlusion and reperfusion (MCAO-R) in the rat. MCAO was induced using the filament method. All animals were subjected to 4 h of MCAO and 24 h of reperfusion. After 24 h, brains were analyzed for size of infarction. Neurological function was assessed during stroke and 24 h after reperfusion. Two groups were studied, an untreated control group (n = 9) and a group treated with the selectin inhibitor, fucoidin (25 mg kg(-1)) (n = 9). We found that selectin blockade significantly reduced cerebral infarction size by 50% (p < 0.05) and improved neurological function (p < 0.05). In addition, a trend toward decreased cerebral edema was demonstrated with selectin inhibition. These results indicate that treatment of the blood and the endothelium with a selectin anti-inflammatory agent is protective after focal stroke and reperfusion in the rat.

Animals↗
PubMed2006

ADAM17 deficiency by mature neutrophils has differential effects on L-selectin shedding.

L-selectin directs neutrophils to sites of inflammation, and upon their activation, surface expression of the receptor is rapidly down-regulated by ectodomain shedding. Tumor necrosis factor-alpha-converting enzyme (TACE, or ADAM17) is a sheddase of L-selectin; however, Adam17 gene targeting (ADAM17(DeltaZn/DeltaZn)) in mice is perinatal lethal and its role in L-selectin shedding by mature neutrophils has not been determined. This was addressed here by using radiation-chimeric mice reconstituted with ADAM17(DeltaZn/DeltaZn) fetal liver cells. ADAM17-deficient neutrophils, monocytes, and lymphocytes failed to shed L-selectin in response to PMA, as did neutrophils infiltrating the inflamed peritoneum. In addition, the absence of functional ADAM17 resulted in significantly increased levels of L-selectin surface expression by peripheral-blood leukocytes, indicating the sheddase also plays a role in the constitutive cleavage of L-selectin. Interestingly, not all manners of L-selectin turnover required ADAM17. Plasma L-selectin levels were similar between ADAM17(DeltaZn/DeltaZn)-chimeric and control mice, as was the shedding of L-selectin by neutrophils undergoing spontaneous apoptosis. The latter process, however, was diminished by a metalloprotease inhibitor, indicating the role of a sheddase other than ADAM17. Together, our data reveal that L-selectin's surface density on neutrophils is regulated by ADAM17, but homeostatic L-selectin cleavage is not.

ADAM Proteins↗
PubMed1996

Effects of estradiol on circulating P-selectin.

The membrane protein P-selectin tethers leukocytes to endothelial cells (EC) and on activated platelets, and may play a role in atherosclerosis. Lower plasma levels of a soluble (c) P-selectin have recently been observed in premenopausal women compared to those in men. Given the antiatherogenic-cardioprotective effect of 17 beta-estradiol (E2), we hypothesized that E2 may down-regulate the expression of P-selectin and subsequently decrease cP-selectin levels. The effects of E2 on levels of plasma cP-selectin were evaluated during the menstrual cycle in healthy women (n = 18) and by measuring the effect of a single im injection of 10 mg E2 valerate (n = 9) or placebo (n = 10) on cP-selectin levels in healthy male volunteers. In women, the cyclic increase in serum E2 concentrations was accompanied by a decrease in cP-selectin levels from 110 ng/mL [95% confidence interval (CI), 100-137 ng/mL] in the follicular phase. The decrease reached a maximum of 13% (95% CI, 2-19%, P = 0.014) in the luteal phase. In men, cP-selectin decreased from a median of 139 ng/mL (95% CI, 113-165) to 125 ng/mL (95% CI, 97-152; P = 0.038) 4 days after E2 injection. The median baseline value of cP-selectin in the 19 male volunteers was 133 ng/mL (95% CI, 115-143 ng/mL) and was approximately 30% higher than those in the female volunteers during the midcycle (P = 0.024) and luteal (P = 0.012) phases, but was not different from that during the follicular phase. In sum, our study suggests that E2 lowers cP-selectin levels. An E2-induced decrease in cP-selectin reflects either decreased activation or damage of platelets and/or endothelial cells in vivo. Thus, our study may point to an additional mechanism for the residual antiatherogenic-cardioprotective effect of E2 that cannot be explained by its lipid-lowering effects.

Adult↗
PubMed1995

Alterations in soluble and leukocyte surface L-selectin (CD 62L) in hemodialysis patients.

Hemodialysis (HD) patients can develop acute reactions during treatment as well as increased long-term susceptibility to infections and malignancy. Leukocyte-membrane interactions may contribute to these processes. The effects of a single HD session on L-selectin, a leukocyte adhesion molecule for endothelium, were examined. Serum levels of soluble L-selectin were measured in 23 patients by enzyme-linked immunosorbent assay before and after a 3-h dialysis session. There was a statistically significant increase in soluble L-selectin from 1.36 +/- 0.12 (SE) to 1.57 +/- 0.18 micrograms/mL (P < 0.001). An increase in shed L-selectin was observed for the "venous" compared with the "arterial" part of the dialyser (P < 0.01) 15 min into HD. Soluble L-selectin levels were found to remain increased at 3 h after treatment. Leukocyte-bound L-selectin and CD11b was examined by the use of flow cytometry. Neutrophil L-selectin decreased to 69 +/- 7% at 15 min (P < 0.01) and then rebounded to 98 +/- 7% at 180 min. Monocyte and lymphocyte L-selectin did not decrease. Because L-selectin is important for leukocyte attachment to endothelium at sites of inflammation, alterations of shed L-selectin and cell-surface L-selectin levels may play a role in the immunologic consequences of HD treatment.

Adult↗
PubMed2003

Lipid raft localization of cell surface E-selectin is required for ligation-induced activation of phospholipase C gamma.

E-selectin, an endothelial cell surface adhesion receptor for leukocytes, also acts as a signaling receptor. Upon multivalent ligation, E-selectin transduces outside-in signals into the endothelium leading to changes in intracellular Ca(2+) concentration and activation of the mitogen-activated protein kinase signaling pathway. In addition, following leukocyte engagement, E-selectin associates via its cytoplasmic domain with components of the actin cytoskeleton and undergoes alterations in phosphorylation state that result in changes in gene expression. In this study, we show that E-selectin is localized in cholesterol-rich lipid rafts at the cell surface, and that upon ligation E-selectin clusters and redistributes in the plasma membrane colocalizing with a fraction of caveolin-1-containing rafts. In addition, we demonstrate that leukocyte adhesion via E-selectin results in association with and activation of phospholipase Cgamma (PLCgamma). Moreover, we show that disruption of lipid rafts with the cholesterol-depleting drug methyl-beta-cyclodextrin disrupts the raft localization of E-selectin as well as the ligation-induced association of E-selectin with PLCgamma, and subsequent tyrosine phosphorylation of PLCgamma. In contrast, cholesterol depletion has no effect on E-selectin-dependent mitogen-activated protein kinase activation. Thus, these findings demonstrate that the presence of E-selectin in lipid rafts is necessary for its association with, and activation of, PLCgamma, and suggest that this subcellular localization of E-selectin is related to its signaling function(s) during leukocyte-endothelial interactions.

Antibodies, Monoclonal↗
PubMed1999

Prognostic value of circulating soluble E-selectin concentrations in node-negative breast cancer patients.

Several studies have suggested that endothelial cells participate in tumor development. Soluble E-selectin (sE-selectin) is specifically released by activated endothelial cells, and its serum concentration can be considered a marker of endothelial activation. In this study, we assessed the prognostic value of sE-selectin concentrations in node-negative breast cancer patients. Serum sE-selectin concentrations were measured by an ELISA method prior to surgery in 456 node-negative breast cancer patients. We analyzed also tumor size (TS), histoprognostic grading, and steroid hormone receptor status. The mean sE-selectin concentration was 24.9 +/- 15.0 ng/ml. The sE-selectin concentrations were mildly correlated with the TS but not with the other factors. For prognostic analyses, the median follow-up duration was 7.5 years. The cutoff sE-selectin concentration used was 40 ng/ml. In overall survival studies, univariate analyses demonstrated a prognostic value of sE-selectin, TS, and histoprognostic grading, and multivariate analyses demonstrated a prognostic value of sE-selectin and TS. For disease-free survival, univariate and multivariate analyses demonstrated a prognostic value of sE-selectin and TS. sE-selectin concentration is an easily measurable and strong prognostic factor in node-negative breast cancer patients. These results provide further evidence for the role of adhesion molecules expression by endothelial cells in tumor progression.

Adult↗
PubMed1999

Loss of endothelial surface expression of E-selectin in a patient with recurrent infections.

Neutrophil accumulation at sites of inflammation is mediated by specific groups of cell adhesion molecules including the beta2 (CD18) integrins on leukocytes and the selectins (P- and E-selectin on the endothelium and L-selectin on the leukocyte). This is supported by studies of patients with leukocyte adhesion deficiency syndromes whose leukocytes are genetically deficient in the expression of beta2 integrins or selectin carbohydrate ligands (eg, sialyl-Lewis(x)). However, inherited deficiency or dysfunction of endothelial cell adhesion molecules involved in leukocyte recruitment has not been previously described. In this report we describe a child with recurrent infections and clinical evidence of impaired pus formation reminiscent of a leukocyte adhesion deficiency syndrome, but whose neutrophils were functionally normal and expressed normal levels of CD18, L-selectin, and sialyl-Lewis(x). In contrast, immunohistochemical staining of inflamed tissue from the patient showed the absence of E-selectin from the endothelium, although E-selectin mRNA was present. However, E-selectin protein was expressed as significantly elevated levels of circulating soluble E-selectin were detected, the molecular size of which was consistent with a proteolytically cleaved form of E-selectin. Gene sequencing failed to show evidence of a secreted mutant variant. These data represent, to our knowledge, the first description of a potentially inherited dysfunction of an endothelial cell adhesion molecule involved in leukocyte recruitment and provide additional human evidence of the importance of endothelial selectins in the inflammatory response.

Bacterial Infections↗