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PubMed2000

Noncovalent association of P-selectin glycoprotein ligand-1 and minimal determinants for binding to P-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1) is a disulfide-bonded, homodimeric mucin ( approximately 250 kDa) on leukocytes that binds to P-selectin on platelets and endothelial cells during the initial steps in inflammation. Because it has been proposed that only covalently dimerized PSGL-1 can bind P-selectin, we investigated the factors controlling dimerization of PSGL-1 and re-examined whether covalent dimers are required for binding its P-selectin. Recombinant forms of PSGL-1 were created in which the single extracellular Cys (Cys(320)) was replaced with either Ser (C320S-PSGL-1) or Ala (C320A-PSGL-1). Both recombinants migrated as monomeric species of approximately 120 kDa under both nonreducing and reducing conditions on SDS-polyacrylamide gel electrophoresis. P-selectin bound similarly to cells expressing either wild type or mutated forms of PSGL-1 in both flow cytometric and rolling adhesion assays. Unexpectedly, chemical cross-linking studies revealed that both C320S- and C320A-PSGL-1 noncovalently associate in the plasma membrane and cross-linking generates dimeric species. Chimeric recombinants of PSGL-1 in which the transmembrane domain in PSGL-1 was replaced with the transmembrane domain of CD43 (CD43TMD-PSGL-1) could not be chemically cross-linked, suggesting that residues within the transmembrane domain of PSGL-1 are required for noncovalent association. Cells expressing CD43TMD-PSGL-1 bound P-selectin. To further address the ability of P-selectin to bind monomeric derivatives of PSGL-1, intact HL-60 cells were trypsin-treated, which generated a soluble approximately 25-kDa NH(2)-terminal fragment of PSGL-1 that bound to immobilized P-selectin. Because N-glycosylation of PSGL-1 hinders trypsin cleavage, a recombinant form of PSGL-1 was generated in which all three potential N-glycosylation sites were mutated (DeltaN-PSGL-1). Cells expressing DeltaN-PSGL-1 bound P-selectin, and trypsin treatment of the cells generated NH(2)-terminal monomeric fragments (<10 kDa) of PSGL-1 that bound to P-selectin. These results demonstrate that Cys(320)-dependent dimerization of PSGL-1 is not required for binding to P-selectin and that a small monomeric fragment of PSGL-1 is sufficient for P-selectin recognition.

Amino Acid Sequence↗
PubMed1997

Tyrosine kinase-dependent regulation of L-selectin expression through the Leu-13 signal transduction molecule: evidence for a protein kinase C-independent mechanism of L-selectin shedding.

The L-selectin adhesion molecule mediates lymphocyte extravasation in peripheral lymph nodes, and has also been implicated in directing leukocyte recruitment to inflammatory tissues and metastasis of lymphoid malignancies. In this study, we demonstrate a novel level of regulation of L-selectin expression that involves the 16-kDa Leu-13 signal transduction molecule. Leu-13 is a member of a multimeric cell surface complex in lymphocytes that includes TAPA-1 (target of antiproliferative Ab-1, CD81) as well as lineage-specific proteins. In the present study, mAb-induced ligation of Leu-13 was shown to rapidly down-regulate L-selectin surface density on normal and malignant human lymphocytes, and to markedly inhibit L-selectin-mediated adhesion of lymphocytes to soluble carbohydrate ligands (i.e., PPME, phosphomonoester core polysaccharide) and to lymph node high endothelial venules. Through the use of genistein and staurosporine, potent inhibitors of tyrosine kinases (TK) and protein kinase C (PKC), respectively, Leu-13-induced L-selectin down-modulation was demonstrated to involve a TK-dependent, PKC-independent pathway, and was attributed to increased L-selectin shedding from surface membranes. Notably, direct L-selectin ligation, modeling cross-linking interactions with endothelial cell ligands, similarly down-regulates L-selectin surface expression through a TK-dependent, PKC-independent mechanism. In sharp contrast, PMA and anti-CD3 mAb down-regulate L-selectin via a staurosporine-sensitive, genistein-resistant pathway that is closely linked to lymphocyte proliferation. Taken together, these results demonstrate a novel role for Leu-13- and L-selectin-induced TK activity in control of L-selectin expression, thus providing insight into the complex molecular mechanisms that potentially regulate L-selectin-dependent lymphocyte homing in vivo.

Antigens, Differentiation↗
PubMed1998

Neutrophil migration into the peritoneum is P-selectin dependent, but sequestration in lungs is selectin independent during peritonitis.

Neutrophil (PMN) influx into the peritoneal cavity in response to bacterial peritonitis is an indispensable aspect of host defense. PMNs also are responsible for the remote organ injury observed after major abdominal infection. The aim of this study was to examine the effect of selectin blockade on PMN migration into the peritoneum and on PMN sequestration in the lungs, early in the course of peritonitis. Cecal ligation and puncture (CLP) was performed on P-selectin-deficient (P-def) mice and their genetic controls (C57). Both groups were treated with anti-E-selectin antibody, anti-L-selectin, or isotypic control immunoglobulin G at the time of CLP. 6 h after CLP, mice were sacrificed. Peritoneal PMN migration decreased in P-def mice compared with C57 controls after CLP. Blockade of E- or L-selectin alone in controls did not alter peritoneal PMN influx or circulating PMNs after CLP. In the P-def mice, treatment with anti-E-antibody or anti-L-antibody nearly eliminated PMN influx into the peritoneum. In contrast, circulating PMNs markedly increased after CLP in P-def mice when compared with baseline values. Lung myeloperoxidase increased in all groups of mice following CLP. Blockade of P-selectin with anti-P-selectin antibody elicited a response similar to that observed in the P-def mice. In conclusion, P-selectin mediates PMN influx into the peritoneal cavity, while E- and L-selectins do not appear to play any significant role in the 6 h time period following CLP. Lung PMN sequestration, after CLP, occurred independent of P-, E-, or L-selectin expression. Blockade of P-selectin during peritonitis appears to be potentially deleterious by preventing early PMN influx into the compartment containing the septic focus.

Animals↗
PubMed2002

L-selectin serves as an E-selectin ligand on cultured human T lymphoblasts.

Previous studies reported that L-selectin (CD62L) on human peripheral blood neutrophils serves as an E-selectin ligand. This study shows that CD62L acquired E-selectin-binding activity following phorbol ester (PMA) treatment of the Jurkat T cell line and anti-CD3/IL-2-driven proliferation of human T lymphocytes in vitro. The recombinant porcine E-selectin/human Ig chimera P11.4 showed neuraminidase-sensitive and calcium-dependent attachment to PMA-stimulated human Jurkat T cells in a flow cytometry assay. The anti-CD62L mAb (DREG 56) blocked this binding interaction by approximately 60% and P11.4 precipitated CD62L from detergent lysates of PMA-activated Jurkat cells. In contrast, P11.4 precipitated minimal amounts of CD62L from detergent lysates of nonactivated human PBL. As reported previously, P-selectin glycoprotein ligand 1 and a distinct 130-kDa glycoprotein were the major species in these precipitates. However, T cell activation on plate-immobilized anti-CD3 and growth in low-dose IL-2 increased the percentage of CD62L molecules with E-selectin-binding activity. After two cycles of activation and culture, approximately 60-70% of the CD62L was precipitated with the P11.4 chimera. These cultured T lymphoblasts rolled avidly on both E-selectin and P-selectin at physiologic levels of linear shear stress. The DREG 56 Ab partially blocked rolling on the E-selectin substrate, whereas no effect was seen on P-selectin. Thus, CD62L on human cultured T lymphoblasts is one of several glycoproteins that interacts directly with E-selectin and contributes to rolling under flow.

Antibodies, Monoclonal↗
PubMed1993

L-selectin can mediate leukocyte rolling in untreated mesenteric venules in vivo independent of E- or P-selectin.

Granulocyte recruitment during the acute inflammatory response is initiated by their rolling along the endothelial lining of postcapillary venules. To determine whether expression of L-selectin alone is sufficient for rolling, the murine pre-B lymphocytic cell line 300.19, which does not bind E- or P-selectin, was transfected with human L-selectin cDNA, which led to stable L-selectin expression at a level similar to that of blood lymphocytes. Fluorescent-labeled cells were infused retrogradely into a side branch of the superior mesenteric artery of anesthetized rats. In venules of the mesenteric membrane, leukocyte rolling occurs without intentional stimulation. On average, 17% +/- 6% of L-selectin transfectants rolled in the observed venules, while mock-transfected cells did not roll. Rolling of L-selectin transfectants began approximately 20 minutes after surgery and continued for at least 120 minutes. In contrast, HL-60 promyelocytes, which express sialyl-Lewis(x) tetrasaccharide (sLe(x)) but not L-selectin and that bind to E- and P-selectin in vitro, did not roll between 20 and 120 minutes, but some HL-60 cells rolled at very early (< 20 minutes) and late (> 2 hours) time points. Pretreatment with either of two function-blocking monoclonal antibodies recognizing the lectin domain of L-selectin completely blocked rolling of L-selectin transfectants and sharply reduced (by 70%) rolling of isolated human granulocytes. Taken together, these results show that L-selectin can mediate leukocyte rolling by virtue of its lectin activity.

Animals↗
PubMed2001

S-100b, sE-selectin, and sP-selectin for evaluation of hypoxic brain damage in patients after cardiopulmonary resuscitation: pilot study.

S-100b is thought to be a screening marker of hypoxic brain damage in patients with cardiac arrest. However, the time-dependent occurrence and relevance of increased S-100b serum levels in out-of-hospital patients with cardiopulmonary resuscitation (CPR) is still discussed. The purpose of our study was to evaluate the diagnostic utility of S-100b measurements in comparison to that of adhesion molecules sE-selectin and sP-selectin in patients with CPR. Sixteen out-of-hospital patients (median age 69.6 years; range 59.2-82.2 years) suffering from cardiac arrest due to ventricular fibrillation, asystole, or electromechanical dissociation were recruited prospectively. Blood samples were drawn on scene after the return of spontaneous circulation (ROSC) and 12 hours after successful CPR. The reference group consisted of 10 patients with isolated severe head trauma (SHT) (Glasgow Coma Score </ or =8), and the control group comprised 20 healthy volunteers. Serum concentrations of S-100b, determined by immunoluminometric assay, were compared with serum levels of sE-selectin and sP-selectin measured by an enzyme-linked immunosorbent assay and correlated with the patients' survival. In the CPR group, S-100b serum levels (2.37 ng/ml; 1.37-4.09 ng/ml) at study entry (11.6 minutes after arriving on scene) did not significantly differ from those of SHT patients (2.88 ng/ml; 1.78-8.81 ng/ml). Both groups showed significant differences from the healthy controls (0.04 ng/ml; 0.01-0.82 ng/ml). At 12 hours after CPR the serum levels had decreased to 0.41 ng/ml (0.24-0.51 ng/ml) but continued to be significantly elevated compared to that of the control group. sE-selectin values in serum increased from 56.00 ng/ml (38.50-85.50 ng/ml) on scene to 79.00 ng/ml (52.00-127.00 ng/ml) after 12 hours (p < 0.05). The first measurements differed significantly from serum levels of the control group (22.50 ng/ml; 14.00-34.00 ng/ml) and from those of the SHT group (45.00 ng/ml; 39.00-63.75 ng/ml). At 12 hours after study entry the sE-selectin values were not significantly different from those of the SHT group (51.50 ng/ml; 39.00-95.88 ng/ml). sP-selectin serum levels increased slightly from 199.50 ng/ml (184.25-227.25 ng/ml) to 247.00 ng/ml (206.50-354.75 ng/ml). First and second measurements did not reveal any significant differences in either the SHT group or the healthy controls. When correlated with survival, S-100b measurements exhibited constantly high serum levels for patients, decreasing within the first 24 hours, whereas they decreased significantly in patients with longer survival. sP-selectin values on scene slightly increased in cases of survivals less than 24 hours after CPR. sE-selectin serum levels always remained within normal levels and revealed no significance later on. In contrast to the endothelium-derived adhesion molecules sE-selectin and sP-selectin, comparison of measurements of specific neuroprotein S-100b early after cardiac arrest and 12 hours later seem to provide an indication of the severity of hypoxic brain damage and the prognosis after CPR. Further investigations are required to better understand the CPR-related mechanisms of blood-brain barrier damage.

Aged↗
PubMed1999

Tyrosine replacement in P-selectin glycoprotein ligand-1 affects distinct kinetic and mechanical properties of bonds with P- and L-selectin.

Selectins are adhesion molecules that initiate tethering and rolling of leukocytes on the vessel wall. Rolling requires rapid formation and breakage of selectin-ligand bonds that must have mechanical strength to resist premature dissociation by the forces applied in shear flow. P- and L-selectin bind to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1), a mucin on leukocytes. To define determinants on PSGL-1 that contribute to the kinetic and mechanical properties of bonds with selectins, we compared rolling of transfected preB cells expressing P- or L-selectin on transfected cell monolayers expressing wild-type PSGL-1 or PSGL-1 constructs with substitutions in targeted N-terminal residues. Rolling through P- or L-selectin required a Thr or Ser at a specific position on PSGL-1, the attachment site for an essential O-glycan, but required only one of three nearby Tyr residues, which are sites for Tyr-SO(3) formation. The adhesive strengths and numbers of cells rolling through P- or L-selectin were similar on wild-type PSGL-1 and on each of the three PSGL-1 constructs containing only a single Tyr. However, the cells rolled more irregularly on the single-Tyr forms of PSGL-1. Analysis of the lifetimes of transient tethers on limiting densities of PSGL-1 revealed that L-selectin dissociated faster from single-Tyr than wild-type PSGL-1 at all shears examined. In sharp contrast, P-selectin dissociated faster from single-Tyr than wild-type PSGL-1 at higher shear but not at lower shear. Thus, tyrosine replacements in PSGL-1 affect distinct kinetic and mechanical properties of bonds with P- and L-selectin.

Amino Acid Sequence↗
PubMed1995

Influence of sE-selectin and L-selectin on the regulation of cell migration during chronic periodontitis.

Soluble endothelial leukocyte adhesion molecule-1 (sE-selectin) levels in peripheral blood (PB) and gingival capillary blood (GCB) of both healthy donors (HD) and patients with adult periodontitis (AP) were assayed by ELISA. Binding of sE-selectin to polymorphonuclear neutrophils (PMN) from PB, GCB and crevicular fluid (GF), and expression of L-selectin and sialyl-Lewisx (sLex) on these cells were analyzed by immunofluorescence and flow cytometry. No significantly enhanced serum levels of sE-selectin in patients with AP, compared to HD (28 +/- 5 ng/ml vs 19 +/- 3 ng/ml, respectively), and no differences in the concentration of sE-selectin in GCB (16 +/- 1 ng/ml vs 16 +/- 2 ng/ml, respectively) were observed. On PB-PMN no significant differences in the expression of L-selectin and sLex were found and binding of sE-selectin to PB-PMN was comparable between HD and patients with AP. Binding of sE-selectin to GCB-PMN was significantly higher in patients with AP compared to HD (mean channel fluorescence (MCF) = 88.5 +/- 13.2 vs MCF = 24.2 +/- 5.3, respectively). The expression of sLex on GCB-PMN did not differ significantly between the two groups. A significant decrease in the expression of the adhesion molecule L-selectin on GCB-PMNs compared to PB-PMN was found in patients with AP but not in HD. CF-PMN showed decreased expression of both L-selectin and sLex compared to PMN from PB and GCB, both in HD and patients with AP. Taken together, these data suggest that PMN from patients with AP had reduced selectin-mediated adhesive capabilities to inflamed gingival endothelium.

Adult↗
PubMed1997

Regulation of E-selectin, P-selectin, and intercellular adhesion molecule 1 expression in mouse cremaster muscle vasculature.

OBJECTIVE: To investigate the expression of P- and E-selectin and intercellular adhesion molecule 1 (ICAM-1) in the vasculature of mouse cremaster muscles both with and without tumor necrosis factor alpha (TNF-alpha) treatment. METHODS: Mice received injections of monoclonal antibody to P-selectin, E-selectin, or ICAM-1 before fixation to restrict detection to antigen expressed on the endothelial surface. Whole-mount preparations of mouse cremaster muscles were fixed in acetone and stained using biotinylated secondary antibody and peroxidase-conjugated streptavidin. RESULTS: P-selectin is expressed on the endothelial surface of cremaster muscle venules within 10 minutes after exteriorization. Expression increases upon treatment with TNF-alpha (2 hours), reflecting transcriptional regulation of P-selectin expression in situ. The baseline E-selectin expression is patchy and barely detectable but shows a significant upregulation after treatment with TNF-alpha. [CAM-1 is constitutively expressed in unstimulated mouse cremaster venules and slightly upregulated after 2 hours of TNF-alpha treatment. Under baseline conditions, neither E-selectin, P-selectin, nor ICAM-1 is detectable in arterioles or capillaries. After TNF-alpha treatment, arterioles stain faintly for P-selectin, but not E-selectin or ICAM-1. CONCLUSION: The temporal and spatial pattern of expression of P- and E-selectin and ICAM-1 is consistent with the functional role of these molecules in mediating preferential leukocyte rolling and adhesion in mouse cremaster muscle venules.

Animals↗
PubMed2000

P-selectin glycoprotein ligand-1 supports rolling on E- and P-selectin in vivo.

Selectin-dependent rolling is the earliest observable event in the recruitment of leukocytes to inflamed tissues. Several glycoproteins decorated with sialic acid, fucose, and/or sulfate have been shown to bind the selectins. The best-characterized selectin ligand is P-selectin glycoprotein-1 (PSGL-1) that supports P-selectin- dependent rolling in vitro and in vivo. In vitro studies have suggested that PSGL-1 may also be a ligand for E- and L-selectins. To study the in vivo function of PSGL-1, without the influence of other leukocyte proteins, the authors observed the interaction of PSGL-1-coated microspheres in mouse venules stimulated to express P- and/or E-selectin. Microspheres coated with functional recombinant PSGL-1 rolled in surgically stimulated and tumor necrosis factor alpha (TNFalpha)-stimulated mouse venules. P-selectin deficiency or inhibition abolished microsphere rolling in surgically and TNFalpha-stimulated venules, whereas E-selectin deficiency or inhibition increased microsphere rolling velocity in TNFalpha-stimulated venules. The results suggest that P-selectin-PSGL-1 interaction alone is sufficient to mediate rolling in vivo and that E-selectin-PSGL-1 interaction supports slow rolling.

Animals↗
PubMed2001

[P-selectin and E-selectin in serum of patients with coronary artery disease].

UNLABELLED: Evidence for the role of chronic inflammation in atherogenesis has been well documented. Selectins mediate the first step in leukocyte adhesion and may contribute to the pathogenesis of stable and unstable angina. METHODS: The study group consisted of 59 patients (pts) with coronary artery disease (CAD) documented coronarographically: 27 pts with stable exertional angina (group A), 32 pts with unstable angina (group B). 20 healthy persons were the control group (group C). Serum levels of E-selectin and P-selectin were measured by ELISA method both before and after the treadmill ECG stress test (ST) in groups A and C. In group B the measurements were carried out at 6, 24, and 48 hours following an episode of chest pain. RESULTS: There were no differences between the baseline serum levels of selectins as determined in groups A and C. In patients with stable angina, the post-ST concentrations of E-selectin were significantly higher (68.8 +/- 29 ng/ml) in comparison to both baseline (38.7 +/- 15 ng/ml), and group C-values (pre-ST: 35.1 +/- 16; post-ST: 49.9 +/- 15 ng/ml). In unstable patients, serum P-selectin levels were higher when compared to those found in groups A and C (group A: 142.3 +/- 24; group B: 190.1 +/- 99; group K: 136.4 +/- 33 ng/ml). No differences between selectins concentrations were observed at fixed times after an episode of chest pain. CONCLUSIONS: Soluble selectins levels in pts with stable angina are comparable to those of healthy persons. Significant increase of E-selectin concentration as induced by ST may reflect endothelial response to exercise. Patients with unstable angina had elevated levels of P-selectin, which seems to be associated with enhanced platelet and leukocyte activation. The serum levels of selectins may indirectly reflect clinical condition of pts with CAD.

Aged↗
PubMed1996

Post-translational modifications of recombinant P-selectin glycoprotein ligand-1 required for binding to P- and E-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like ligand for P- and E-selectin on human leukocytes. PSGL-1 requires sialylated, fucosylated O-linked glycans and tyrosine sulfate to bind P-selectin. Less is known about the determinants that PSGL-1 requires to bind E-selectin. To further define the modifications required for PSGL-1 to bind P- and E-selectin, we transfected Chinese hamster ovary (CHO) cells with cDNAs for PSGL-1 and specific glycosyltransferases. CHO cells synthesize only core 1 O-linked glycans (Galbeta1-3GalNAcalpha1-Se r/Thr); they lack core 2 O-linked glycans (Galbeta1-3(Galbeta1-4GlcNAcbeta1-6)GalNAcalpha1 -Ser/Thr) because they do not express the core 2 beta1 6-N-acetylglucosaminyltransferase (C2GnT). CHO cells also lack alpha1 3 fucosyltransferase activity. PSGL-1 expressed on transfected CHO cells bound P- and E-selectin only when it was co-expressed with both C2GnT and an alpha1 3 fucosyltransferase (Fuc-TIII, Fuc-TIV, or Fuc-TVII). Chromatography of beta-eliminated O-linked glycans from PSGL-1 co-expressed with C2GnT confirmed synthesis of core 2 structures. Tyrosine residues on PSGL-1 expressed in CHO cells were shown to be sulfated. Phenylalanine replacement of three tyrosines within a consensus sequence for tyrosine sulfation abolished binding to P-selectin but not to E-selectin. These results demonstrate that PSGL-1 requires core 2 O-linked glycans that are sialylated and fucosylated to bind P- and E-selectin. PSGL-1 also requires tyrosine sulfate to bind P-selectin but not E-selectin.

Animals↗
PubMed1998

A novel P-selectin glycoprotein ligand-1 monoclonal antibody recognizes an epitope within the tyrosine sulfate motif of human PSGL-1 and blocks recognition of both P- and L-selectin.

Interactions between P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) mediate the earliest "rolling" of leukocytes on the lumenal surface of endothelial cells at sites of inflammation. Previously, PSGL-1 has been shown to be the primary mediator of interactions between neutrophils and P-selectin, but studies on the ability of PSGL-1 to mediate interactions between P-selectin and other subsets of leukocytes have yielded variable and conflicting results. A novel IgG monoclonal antibody (MoAb) to human PSGL-1 was generated, and the specificity of this MoAb was confirmed by both flow cytometric analysis and Western blotting of cells transfected with human PSGL-1. This newly developed MoAb, KPL1, inhibited interactions between P-selectin expressing COS cells and either HL60 cells, neutrophils, or lymphocytes. Furthermore, KPL1 completely inhibited interactions between P-selectin and either purified CD4 T cells or neutrophils in a flow assay under physiological conditions, but had no effect on interactions of T cells or neutrophils with E-selectin. In addition, KPL1 blocked interactions between lymphoid cells transfected with L-selectin and COS cells expressing PSGL-1. The KPL1 epitope was mapped to a site within a consensus tyrosine sulfation motif of PSGL-1, previously shown to be essential for interaction with P-selectin and now shown to be essential for interaction with L-selectin, and to be distinct from the epitope identified by the PL1 function blocking anti-PSGL-1 MoAb. Two-color flow cytometry of normal leukocytes showed that while natural killer (NK) cells (CD16(+)), monocytes, CD4 and CD8 T cells, and alpha/beta and gamma/delta T cells were uniformly positive for PSGL-1, B cells expressed low levels of the KPL1 epitope. This low level of KPL1 staining was also observed immunohistologically in germinal centers, which had no detectable KPL1 staining, whereas T-cell areas (interfollicular region) were positive for KPL1. Interestingly, plasma cells in situ and interleukin-6-dependent myeloma cell lines were KPL1(+). Thus, PSGL-1 is expressed on essentially all blood neutrophils, NK cells, B cells, T cells, and monocytes. Variation in tyrosine sulfation during B-cell differentiation may affect the ability of B cells to interact with P- and L-selectin.

Animals↗
PubMed2000

Expression of platelet P-selectin and detection of soluble P-selectin, NPY and RANTES in patients with inflammatory bowel disease.

OBJECTIVE AND DESIGN: P-selectin, a membrane glycoprotein which is expressed on activated platelets and endothelial cells, plays a crucial role in the inflammatory response. The main action is adhesion of leukocytes, facilitation of diapedesis and induction of cytokine production from monocytes (MCP-1 and IL-8), mediated via RANTES released from activated platelets. An abnormal platelet activity has been reported in patients with ulcerative colitis (UC) and Crohn's disease (CD), jointly referred to as inflammatory bowel disease (IBD), which could have an aggravating influence on the inflammatory response. In addition, an up-regulation of platelet IL-8 receptors among patients with IBD has been reported. To reveal a presumptuous platelet dysfunction we analysed the expression of platelet surface P-selectin at resting state and after stimulation with thrombin, collagen, epinephrine and interleukin 8 (IL-8), and plasma levels of soluble P-selectin, neuropeptide Y (NPY) and RANTES in patients with IBD. SUBJECTS: Blood from twelve healthy subjects (control group) and twenty-one patients with IBD who had not taken any anti-platelet drugs or steroids were analysed. METHODS: Patients were sub-grouped according to disease entity, disease activity and 5ASA medication. Surface P-selectin expression on isolated human platelets and plasma P-selectin, NPY and RANTES were analysed with ELISA. All values are presented as mean +/- standard error of the mean (SEM). Mann-Whitney U test and Wilcoxon matched rank test were used for statistical analyses. RESULTS: Patients with IBD in remission (n = 9) had higher basal P-selectin expression, 0.38+/-0.04, compared to the control group (n = 12), 0.22+/-0.03,p < 0.01. UC patients (n = 16) showed down-regulation of P-selectin expression after stimulation with IL-8, 0.26+/-0.03 to 0.22+/-0.02, p < 0.05. No significant differences could be observed concerning soluble P-selectin and NPY in plasma. Patients with 5ASA (n = 12) had lower levels of plasma RANTES, 2.39+/-0.06 microg/l, compared to the control group (n = 12), 3.29+/-0.19 microg/l, p < 0.01, and patients without 5ASA (n = 9), 2.90+/-0.17 microg/l, p < 0.05. CONCLUSIONS: Patients with IBD in remission have higher basal platelet surface P-selectin expression. An exaggerated platelet activity with increased expression of platelet P-selectin and release of inflammatory mediators such as RANTES, which is chemotactic and induce chemokine production, could have a reinforcing and aggravating influence on the inflammatory response and increase the susceptibility to IBD. In addition IL-8 has a down-regulating effect on platelet surface P-selectin expression and 5ASA medication seems to lower plasma RANTES. If 5ASA is responsible for lowering the concentration of RANTES this could be one of the beneficial outcomes of 5ASA medication.

Adult↗
PubMed2002

Ultrastructural examination of cytoskeletal linkage of L-selectin and comparison of L-selectin cytoskeletal association to that of other human and bovine lymphocyte surface antigens.

L-selectin is constitutively expressed on most leukocytes and is responsible for the initial events in cell trafficking termed tethering and rolling. Recently, L-selectin has been shown to associate with the actin-based cytoskeleton under a variety of conditions. In an effort to better understand L-selectin cytoskeletal association and the ultrastructural nature of the cytoskeleton itself, we provide a comparison of the cytoskeletal association of various human and bovine surface proteins in relation to L-selectin. Electron microscopic examination of the cytoskeleton provided further data on the ultrastructure of freshly isolated peripheral lymphocytes as well as demonstrated L-selectin localization to the periphery of the cytoskeleton following low dose detergent treatment of the cell. Clusters of colloidal-gold-stained L-selectin were found on the surface of the detergent-treated lymphocytes, even though these particles completely lacked microvilli. By flow cytometry, we have defined three distinct patterns of cytoskeletal association; constitutive, inductive, and mAb crosslink-induced, and assigned human and bovine CD2, CD3, CD4, CD5, CD8, CD18, CD19, CD44, CD45RA, CD45RO, alphabeta TCR, gammadelta TCR, E-selectin ligands, and L-selectin surface antigens to one of these respective patterns. SDS-PAGE analyses confirmed most of the flow cytometry results. Depending upon its conformation, L-selectin fell into the inductive or mAb crosslink-induced pattern of association, similar to E-selectin ligand(s). Our data provide additional insight into the functional role of L-selectin and the cytoskeleton in immunological events.

Animals↗
PubMed1998

Neutrophil cathepsin G modulates platelet P-selectin expression and inhibits P-selectin-mediated platelet-neutrophil adhesion.

1. Close contact between platelets and neutrophils modulates their cellular interactions in thrombotic and inflammatory states, with stimulation of P-selectin expression on platelets by agonists such as thrombin and neutrophil-derived cathepsin G being critical in mediating platelet-neutrophil adhesion. This study compared the effects of thrombin and cathepsin G on platelet P-selectin expression and on P-selectin-mediated platelet-neutrophil adhesion. 2. Washed platelets and platelet-neutrophil mixed cell suspensions (platelet/neutrophil ratio, 10:1) were incubated with either the supernatant of activated neutrophils, purified cathepsin G or thrombin. Platelet P-selectin expression and platelet adhesion to neutrophils was quantified by flow fluorocytometric analysis. 3. The supernatant from activated neutrophils stimulated platelet P-selectin expression comparable to that produced by purified cathepsin G or thrombin. P-selectin expression induced by both activated neutrophil supernatant and purified cathepsin G was completely inhibited by alpha 1-antichymotrypsin, a specific inhibitor of cathepsin G. Unlike thrombin, which induced maximum platelet P-selectin expression by 10 min, sustained to 120 min, cathepsin G induced an initial large increase in platelet P-selectin expression, followed by a progressive reduction over 30-60 min to baseline levels. 4. Co-incubation of neutrophils with thrombin-stimulated platelets resulted in a significant increase in P-selectin-mediated platelet-neutrophil adhesion, which was completely inhibited by preincubation of neutrophils with anti-sialyl Lewis(x) monoclonal antibody. Thrombin produced maximum platelet-neutrophil adhesion by 10 min which remained stable over 120 min. In contrast, cathepsin G-stimulated platelets did not adhere to neutrophils over 120 min of co-incubation. Addition of cathepsin G to thrombin-stimulated platelets caused a progressive reduction over 30-60 min to baseline levels of platelet-neutrophil adhesion. 5. Neutrophil-derived cathepsin G is a potent platelet activator, but unlike thrombin it causes a time-dependent loss of platelet P-selectin expression and inhibits P-selectin-mediated platelet-neutrophil adhesion. Therefore, cathepsin G may modulate thrombin-mediated platelet-neutrophil adhesive interactions in inflammation and thrombosis.

Adult↗
PubMed2000

Decreased expression levels of L-selectin on subsets of leucocytes and increased serum L-selectin in severe psoriasis.

L-selectin is a leucocyte adhesion molecule involved in leucocyte interactions with vascular endothelial cells. Following leucocyte activation L-selectin is endoproteolytically released from the cell surface. To assess whether psoriasis vulgaris results in systemic leucocyte activation, we examined expression levels of L-selectin on subsets of peripheral blood leucocytes from patients with psoriasis (n = 25) and normal control subjects. Serum levels of soluble L-selectin were quantified by ELISA in patients with psoriasis (n = 75), pustulosis palmaris et plantaris, and contact dermatitis, as well as normal control subjects. Psoriasis severity was evaluated by psoriasis area and severity index (PASI). L-selectin expression levels on CD4+ T cells, B cells, monocytes, and neutrophils from patients with severe-type psoriasis (PASI > or = 15) was significantly decreased compared with leucocytes from normal control subjects. Furthermore, L-selectin expression on CD4+ T cells showed good inverse correlation with PASI scores. Monocyte L-selectin expression was restored when the skin lesions of psoriasis were remitted. The frequencies of L-selectin+ CD4+ T cells or L-selectin+ CD8+ T cells from patients with psoriasis were almost normal. Serum L-selectin levels in patients with severe-type psoriasis were significantly higher than those in normal control subjects. These results suggest that subsets of leucocytes may be activated in psoriasis, and that L-selectin expression levels on some leucocyte subsets, especially CD4+ T cells, tend to correlate with disease severity of psoriasis.

Adult↗
PubMed2001

Affinity, kinetics, and thermodynamics of E-selectin binding to E-selectin ligand-1.

E-selectin is an endothelial adhesion molecule, which mediates the tethering and rolling of leukocytes on vascular endothelium. It recognizes the glycoprotein E-selectin ligand-1 (ESL-1) as a major binding partner on mouse myeloid cells. Using surface plasmon resonance, we measured the kinetics and affinity of binding of monomeric E-selectin to ESL-1 isolated from mouse bone marrow cells. E-selectin bound to ESL-1 with a fast dissociation rate constant of 4.6 s(-1) and a calculated association rate constant of 7.4 x 10(4) m(-1) s(-1). We determined a dissociation constant (K(d)) of 62 microm, which resembles the affinity of L-selectin binding to glycosylation-dependent cell adhesion molecule-1. The affinity of the E-selectin-ESL-1 interaction did not change significantly when the temperature was varied from 5 degrees C to 37 degrees C, indicating that the enthalpic contribution to the binding is small at physiological temperatures, and that, in contrast to typical protein-carbohydrate interactions, binding is driven primarily by favorable entropic changes. Interestingly, surface plasmon resonance experiments with recombinant ESL-1 from alpha 1,3-fucosyltransferase IV-expressing Chinese hamster ovary cells showed a very similar K(d) of 66 microm, suggesting that this fucosyltransferase is sufficient to produce fully functional recombinant ESL-1. Following the recent description of the affinity and kinetics of the selectin-ligand pairs L-selectin-glycosylation-dependent cell adhesion molecule-1 and P-selectin-P-selectin glycoprotein ligand-1, this is the first determination of the parameters of E-selectin binding to one of its naturally occurring ligands.

Animals↗