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.     

Recombinant P-selectin glycoprotein ligand-1 directly inhibits leukocyte rolling by all 3 selectins in vivo: complete inhibition of rolling is not required for anti-inflammatory effect

Selectin-dependent leukocyte rolling is one of the earliest steps of an acute inflammatory response and, as such, contributes to many inflammatory diseases. Although inhibiting leukocyte rolling with selectin antagonists is a strategy that promises far-reaching clinical benefit, the perceived value of this strategy has been limited by studies using inactive, weak, or poorly characterized antagonists. Recombinant P-selectin glycoprotein ligand-1-immunoglobulin (rPSGL-Ig) is a recombinant form of the best-characterized selectin ligand (PSGL-1) fused to IgG, and is one of the best prospects in the search for effective selectin antagonists. We have used intravital microscopy to investigate the ability of rPSGL-Ig to influence leukocyte rolling in living blood vessels and find that it can reduce rolling dependent on each of the selectins in vivo. Interestingly, doses of rPSGL-Ig required to reverse pre-existing leukocyte rolling are 30-fold higher than those required to limit inflammation, suggesting additional properties of this molecule. In support of this, we find that rPSGL-Ig can bind the murine chemokine KC and inhibit neutrophil migration toward this chemoattractant in vitro.     

An L-selectin ligand distinct from P-selectin glycoprotein ligand-1 is expressed on endothelial cells and promotes neutrophil rolling in inflammation

Neutrophils recruited from the blood are key players in the innate immune response. Selectins play critical roles in neutrophil recruitment by mediating their tethering and rolling in inflamed venules. While the roles of P- and E-selectin in this process are well established, the mechanisms of L-selectin-mediated neutrophil recruitment remain elusive. One proposal is that tethering is mediated by L-selectin on flowing neutrophils interacting with P-selectin glycoprotein ligand-1 (PSGL-1) on adherent neutrophils. To clarify whether L-selectin-mediated neutrophil recruitment depends entirely on PSGL-1, we examined the impact of L-selectin deficiency in mice with a PSGL-1-deficient background. L-selectin and PSGL-1 double-knockout mice exhibited a higher increase in their peripheral blood neutrophil count and a worse defect in neutrophil recruitment into the inflamed peritoneum than PSGL-1-deficient mice. Intravital microscopy of inflamed cremaster muscle venules showed that L-selectindeficiency or antibody blockade of L-selectin reduced the residual leukocyte rolling in PSGL-1-deficient mice. Flow cytometric analyses showed that the endothelial cells from the cremaster muscle bound L-selectin in a PSGL-1-independent manner. These results provide evidence for the existence of an L-selectin ligand distinct from PSGL-1 in inflammation and indicate that such a ligand is expressed on endothelial cells, promoting neutrophil rolling in vivo.     

Core2 beta-1,6-N-acetylglucosaminyltransferase enzyme activity is critical for P-selectin glycoprotein ligand-1 binding to P-selectin

P-selectin glycoprotein ligand-1 (PSGL-1) is a high-affinity counterreceptor for P-selectin on myeloid cells and activated T-cells. In addition, PSGL-1 can serve, both in vitro and in vivo, as an E- selectin ligand. Appropriate glycosylation of PSGL-1 is crucial for binding to P-selectin. Functional PSGL-1 is known to bear sialyl lewis X (SLex) or a closely related oligosaccharide. In this study, we show that Chinese hamster ovary (CHO) cells expressing PSGL-1 and fucosyltransferase show a dramatic increase in binding to P-selectin when transfected with "core2" transferase, the enzyme that initiates branching of O-linked glycans. Moreover, only PSGL-1 from core2 transfectant CHO cells can be affinity-captured with P-selectin, suggesting that branched O-linked glycans are required for high- affinity binding to P-selectin. Analysis of PSGL-1-derived O-linked oligosaccharides produced in core2 transfected cells shows the presence of more elaborated glycans. Interestingly, transfection of core2 in these cells does not alter binding to E-selectin.     

N-terminal residues in murine P-selectin glycoprotein ligand-1 required for binding to murine P-selectin

P-selectin binds to the N-terminal region of human P-selectin glycoprotein ligand-1 (PSGL-1). For optimal binding, this region requires sulfation on 3 tyrosines and specific core-2 O-glycosylation on a threonine. P-selectin is also thought to bind to the N terminus of murine PSGL-1, although it has a very different amino acid sequence than human PSGL-1. Murine PSGL-1 has potential sites for sulfation at Tyr13 and Tyr15 and for O-glycosylation at Thr14 and Thr17. We expressed murine PSGL-1 or constructs with substitutions of these residues in transfected Chinese hamster ovary cells that coexpressed the glycosyltransferases required for binding to P-selectin. The cells were assayed for binding to fluid-phase P-selectin and for tethering and rolling on P-selectin under flow. In both assays, substitution of Tyr13 or Thr17 markedly diminished, but did not eliminate, binding to P-selectin. In contrast, substitution of Tyr15 or Thr14 did not affect binding. Substitution of all 4 residues eliminated binding. Treatment of cells with chlorate, an inhibitor of sulfation, markedly reduced binding of wild-type PSGL-1 to P-selectin but did not further decrease binding of PSGL-1 with substitutions of both tyrosines. These data suggest that sulfation of Tyr13 and O-glycosylation of Thr17 are necessary for murine PSGL-1 to bind optimally to P-selectin. Because it uses only one tyrosine, murine PSGL-1 may rely more on other peptide components and O-glycosylation to bind to P-selectin than does human PSGL-1.     

P-selectin glycoprotein ligand-1 (PSGL-1) is a ligand for L-selectin in neutrophil aggregation

In inflammation, activated neutrophils adhere to endothelial cells and aggregate with one another. While beta 2-integrin and L-selectin are essential for aggregation, their ligands remain to be identified. We have previously shown that L-selectin mediates a carbohydrate-dependent interaction in aggregation (Simon et al: J Immunol 149:2765, 1992; Rochon et al: J Immunol 152:1385, 1994). We have suggested that the L- selectin counter-structure is a mucinlike protein and proposed that aggregation occurs through a two-step process involving L-selectin, beta 2-integrin, and their distinct counter-structures (Bennett et al: J Leuk Biol 58:510, 1995). A candidate ligand for L-selectin is P- selectin glycoprotein ligand-1 (PSGL-1), a mucinlike protein on neutrophils that binds P-and E-selectin. Using flow cytometry we show that the number and size of neutrophil aggregates is reduced with Fab fragments of PL1, an anti-PSGL-1 monoclonal antibody that blocks the interaction between P-selectin and PSGL-1 (Moore et al: J Cell Biol 128:661, 1995). In addition, monoclonal antibodies to L-selectin and PSGL-1 were used simultaneously to modulate the availability of these adhesion molecules on individual cell populations. The inhibition of aggregation by these antibodies is consistent with L-selectin and PSGL- 1 being counter-structures. We suggest that L-selectin and PSGL-1 support a collisional cell-cell interaction that represents the first step in neutrophil aggregation.     

Regulation of P-selectin binding to the neutrophil P-selectin counter-receptor P-selectin glycoprotein ligand-1 by neutrophil elastase and cathepsin G

In the inflammatory response, leukocyte rolling before adhesion and transmigration through the blood vessel wall is mediated by specific cell surface adhesion receptors. Neutrophil rolling involves the interaction of P-selectin expressed on activated endothelium and its counter-receptor on neutrophils, P-selectin glycoprotein ligand-1 (PSGL-1). Here, it is reported that P-selectin binding to neutrophils is lost under conditions that cause the release of proteinases from neutrophil primary granules. Treatment of neutrophils with the purified neutrophil granule proteinases, cathepsin G and elastase, rapidly abolished their capacity to bind P-selectin. This inactivation corresponded to loss of the N-terminal domain of PSGL-1, as assessed by Western blot analysis. A loss of intact PSGL-1 protein from the surfaces of neutrophils after the induction of degranulation was also detected by Western blot analysis. Cathepsin G initially cleaved near the PSGL-1 N-terminus, whereas neutrophil elastase predominantly cleaved at a more C-terminal site within the protein mucin core. Consistent with this, cathepsin G cleaved a synthetic peptide based on the PSGL-1 N-terminus between Tyr-7/Leu-8. Under conditions producing neutrophil degranulation in incubations containing mixtures of platelets and neutrophils, the loss of PSGL-1, but not P-selectin, from platelet-neutrophil lysates was detected. Cathepsin G- or neutrophil elastase-mediated PSGL-1 proteolysis may constitute a potential autocrine mechanism for down-regulation of neutrophil adhesion to P-selectin.     

Cross-linking of P-selectin glycoprotein ligand-1 induces death of activated T cells

Increasing evidence has shown that death signaling in T cells is regulated in a complicated way. Molecules other than death receptors can also trigger T-cell death. Here, we demonstrate for the first time that P-selectin glycoprotein ligand-1 (PSGL-1) or CD162 molecules cross-linked by an anti-PSGL-1 monoclonal antibody, TAB4, can trigger a death signal in activated T cells. In contrast to classic cell death, PSGL-1-mediated T-cell death is caspase independent. It involves translocation of apoptosis-inducing factor from mitochondria to nucleus and mitochondrial cytochrome c release. Ultrastructurally, both peripheral condensation of chromatin and apoptotic body were observed in PSGL-1-mediated T-cell death. Collectively, this study demonstrates a novel role for PSGL-1 in controlling activated T-cell death and, thus, advances our understanding of immune regulation.     

Short alleles of P-selectin glycoprotein ligand-1 protect against premature myocardial infarction

Background

Atherosclerosis is an inflammatory disease resulting from an injury that leads to an increase in the adhesiveness and permeability of the endothelium to leukocytes or platelets. The selectin family plays a key role initiating the cascade of events. Recently, we have demonstrated the functional relevance of a variable number of tandem repeats polymorphism affecting the P-selectin glycoprotein ligand (PSGL-1). Neutrophils carrying short alleles exhibit a significantly lower capacity to bind activated platelets. These alleles consistently protect against transient ischemic attack. We sought to evaluate the role of this polymorphism in premature myocardial infarction because genetic risk factors have more relevance in the development of disease in young patients.

Methods

We genotyped 219 Caucasian patients who had suffered a premature myocardial infarction (MI) (aged ≤45 years) and 594 control subjects from our Mediterranean area. The role of the PSGL-1 polymorphism was also evaluated according to the additional risk factors of age, sex, smoking history, hypertension, hypercholesterolemia, and diabetes.

Results

The frequency of the short alleles (B and C) was significantly lower in patients than in controls (P = .012, odds ratio 0.62; 95% CI 0.42-0.92). Multiple regression analysis revealed that B and C alleles had an independent protective effect on the development of premature MI (P = .034, odds ratio 0.62 95%CI: 0.40-0.96).

Conclusions

We found an interesting association between a functional polymorphism and the risk of MI at a younger age. According to our results, the short B and C PSGL-1 alleles might protect against premature MI, probably because of their lesser adhesive capacity.     

P-selectin glycoprotein ligand-1 VNTR polymorphisms and risk of thrombosis in the antiphospholipid syndrome

OBJECTIVES: Antiphospholipid antibodies (aPLA) have been shown to enhance thrombus formation by increasing the expression of adhesive receptors such as P-selectin on endothelial cells. The P-selectin counter-receptor on leucocytes is P-selectin glycoprotein ligand-1 (PSGL-1). We have previously described a variable number of tandem repeats (VNTR) polymorphism in the mucin-like region of PSGL-1, with three alleles: allele A, 16 repeats; allele B, 15 repeats; and allele C, 14 repeats. METHODS: We compared the PSGL-1 VNTR allele and genotype frequencies in 90 patients with antiphospholipid syndrome (APS) with thrombosis, 39 patients with persistent aPLA positivity without thrombosis, and 203 healthy controls. RESULTS: The frequency of the B allele was significantly higher in patients with APS with thrombosis compared with patients without thrombosis (p = 0.023). When we compared the groups by genotype frequencies, we found a markedly higher frequency of the AB genotype in patients with APS with thrombosis than in aPLA-positive patients without thrombosis (38.9% vs 10.3%, p = 0.001) or in normal population (38.9% vs 22.2%, p<0.01). CONCLUSIONS: We suggest that the VNTR polymorphism of PSGL-1 is a significant determinant of thrombotic predisposition in patients with APS. Furthermore, risk appears to correlate best with the combination of alleles inherited rather than with the presence of any particular allele.     

The association of P-selectin glycoprotein ligand-1 VNTR polymorphisms with coronary stent restenosis

Background P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) regulate the initial interactions between leukocytes, activated platelets and endothelial cells. Recently, a variable number of tandem repeats (VNTR) polymorphism in PSGL-1 gene affecting the length of the extracellular domain of PSGL-1 and the distance of the P-selectin binding site to the cell surface has been described. There are limited numbers of studies reporting PSGL-1 polymorphism might affect the inflammatory response and thrombosis. We explored the association between PSGL-1 VNTR polymorphisms (especially AB genotype that has the most deformed configuration of the binding site) and the development of coronary stent restenosis and stent thrombosis in patients with coronary artery disease (CAD). Materials and methods Eighty-seven patients with in-stent restenosis and 93 patients with patent coronary stents were included into the study. The distributions of age, gender, hypertension, diabetes, smoking, total cholesterol and triglyceride levels were similar between the groups. Genomic DNAs were obtained by standard methods from whole blood samples. Specific primers were used to amplify PSGL-1 gene by polymerase chain reaction (PCR). Results Three alleles and 5 different genotypes were detected. In the in-stent restenosis group; allele frequencies were 79.5% for A allele, 18.1% for B allele and 2.4% for C allele and in the patent stent group; allele frequencies were 79.3% for A allele, 20.1% for B allele and 0.6% for C allele. The allele frequencies were similar between the in-stent restenosis group and patent stent group (P = 0.97 for A allele, P = 0.73 for B allele and P = 0.19 for C allele). Genotype distributions were also similar between the groups. There were not any significant associations between PSGL-1 AB genotype and stent restenosis (31.3% vs. 27.2%, P = 0.54), repetitive stent restenosis (33.3% vs. 28.8%, P = 0.82) or in-stent thrombosis (44.4% vs. 28.2%, P = 0.37). In neither male patients nor female patients, there was any significant association between AB genotype and restenosis (32.2% vs. 24.3% P = 0.31 and 29.2% vs. 38.9% P = 0.51, respectively). However, among patients with a family history of early CAD, significantly higher percentage of AB genotype was found in those with stent restenosis (41.4% vs. 18.8%, P = 0.03). Conclusions No significant association was found between PSGL-1 VNTR polymorphisms and in-stent restenosis. However, in patients with a family history of early CAD presence of PSGL-1 AB genotype might increase the risk of in-stent restenosis.     

Identification of P-selectin glycoprotein ligand-1 as a useful marker in acute myeloid leukaemias

Immunophenotyping is considered to be less valuable in the diagnosis of acute myeloid leukaemias (AML) compared with acute lymphoid leukaemias. Here, we present data on the use of quantitative flow cytometry (QFC) of P-selectin glycoprotein ligand 1 (PSGL-1, CD162) and three-colour immunophenotyping including CD162 staining in the identification of myeloid precursors in AML. Analysis of normal peripheral blood (n = 20) and normal bone marrow (n = 5) samples and on 20 samples from de novo M1, M2, M4 and M5 AML patients demonstrated that PSGL-1 is differentially expressed on various mature and immature leucocyte subsets. It was found by QFC that neutrophils expressed 26500 +/- 4500 and monocytes 47200 +/- 9900 copies of PSGL-1 on their surface, whereas AML blasts from M1 and M2 AML patients expressed significantly less PSGL-1 (12 000 +/- 5300) than mature neutrophils (P < 0.001). In M4 and M5 leukaemias, however, the amount of PSGL-1 on monocytic precursors is displayed in a fairly broad range that is not significantly different from that of mature monocytes (P = 0.084). Using three-colour immunophenotyping PSGL-1-dim staining was co-expressed with CD7 and C34 positivity and PSGL-1 staining intensity on immature myeloid cells paralleled with CD45 expression. This would imply a differential expression of PSGL-1 during myeloid haematopoietic development and suggests that quantification of surface PSGL-1 may aid in differentiating myeloblasts from monoblasts by immunophenotyping in different AML subsets.     

Recombinant soluble P-selectin glycoprotein ligand-1 protects against myocardial ischemic reperfusion injury in cats.

OBJECTIVE: Neutrophils (PMNs) contribute importantly to the tissue injury associated with ischemia and subsequent reperfusion of a vascular bed. The effects of a recombinant soluble human form of P-selectin glycoprotein ligand-1 (rsPSGL.Ig) on PMN-endothelial cell interactions were investigated in a well established model of feline myocardial-ischemia reperfusion injury. METHODS: Cats were subjected to 90 min of myocardial ischemia followed by 270 min of reperfusion. RESULTS: Administration of rsPSGL.Ig (1 mg/kg) just prior to reperfusion resulted in a significant reduction in myocardial necrosis compared to that in cats administered a low affinity mutant form of rsPSGL.Ig (1 mg/kg) (16 +/- 3 vs. 42 +/- 7% of area-at-risk, P < 0.01). Cardioprotective effects were confirmed by significant (P < 0.05) reductions in plasma creatine kinase activity in cats treated with rsPSGL.Ig. Inhibition of PMN-endothelial cell interactions was evidenced by a significant attenuation in cardiac myeloperoxidase activity (P < 0.01) and reduced PMN adherence to ischemic-reperfused coronary endothelium (P < 0.001). In addition, rsPSGL.Ig treatment significantly (P < 0.01) preserved endothelium-dependent vasorelaxation in ischemic-reperfused coronary arteries. CONCLUSION: These results demonstrate that the administration of a recombinant soluble PSGL-1 reduces myocardial reperfusion injury and preserves vascular endothelial function, which is largely the result of reduced PMN-endothelial cell interactions.     

Mouse P-selectin glycoprotein ligand-1: molecular cloning, chromosomal localization, and expression of a functional P-selectin receptor

A mouse homolog of P-selectin glycoprotein ligand-1 (PSGL-1), a P- selectin receptor on myeloid cells, has been cloned using the human cDNA sequence to probe a cDNA library prepared from the mouse WEHI-3 monocytic cell line and a genomic DNA library prepared from 129/SvJ mouse tissue. The gene flanking the entire open reading frame of 397 amino acids is composed of a single exon. Mouse and human PSGL-1 show an overall similarity of 67% and an identity of 50% and contain a similar domain organization. However, there are 10 threonine/serine- rich decameric repeats in mouse PSGL-1 as compared with 15 threonine- rich repeats in human PSGL-1. When the mouse PSGL-1 cDNA is coexpressed with an alpha 1,3/1,4 fucosyltransferase cDNA in COS cells, a functional protein is expressed on the COS cell surface mediating binding to human P-selectin. The mouse PSGL-1 gene, Selpl, was mapped to a position on mouse chromosome 5 (Chr 5). Northern blot analyses of mouse tissues showed moderate expression of a PSGL-1 mRNA species in most tissues including heart, kidney, liver, muscle, ovary, and stomach and high levels of expression in blood, bone marrow, brain, adipose tissue, spleen, and thymus. Whereas certain mouse myeloid cell lines including PU5-1.8, WEHI-3B, and 32DC13 express high levels of PSGL-1 mRNA, only WEHI-3B and 32DC13 bind to P-selectin; this interaction is blocked by anti-PSGL-1 antibody. WEHI-3B cells bind significantly better to P-selectin than to E-selectin. Although comparable P-selectin binding is observed in 32DC13 cells, these cells bind better to E- selectin. Binding of 32DC13 cells to E-selectin is not blocked by anti- PSGL-1 antibody. Treatment of WEHI-3B cells with trypsin or neuraminidase abolished their ability to interact with P-selectin. These results indicate that mouse PSGL-1 has structural and functional homology to human PSGL-1 but is characterized by differences in the composition and number of the decameric repeats. PSGL-1 on mouse myeloid cells is critical for high-affinity binding to P-selectin but not E-selectin.     

P-selectin mediates spontaneous leukocyte rolling in vivo

Rolling represents the initial step leading to leukocyte extravasation from blood vessels during an inflammatory reaction. In vitro studies indicate that P-selectin could be one of the ligands on endothelium involved in the rolling phenomenon, although the molecular determinants responsible for this transient attachment in vivo are still undefined. Our objectives were to develop a blocking monoclonal antibody against canine P-selectin and to use it to investigate the role of P-selectin in leukocyte rolling in vivo using the technique of intravital microscopy. P-selectin was immunoaffinity purified from canine platelets and used for the production of monoclonal antibodies. One of the hybridomas generated, MD6, was shown by enzyme-linked immunosorbent assay and by flow cytometry to bind preferentially to stimulated platelets and to completely prevent binding of stimulated platelets to neutrophils. Visualization of canine mesenteric venules by intravital microscopy showed that administration of MD6 resulted in a marked inhibition in the number of rolling leukocytes (18.96 +/- 9.92 v 156.40 +/- 19.50 leukocytes/min, P < .05; 88.3% +/- 6.0% inhibition). Control antibody MD3 (which recognizes a nonfunctional epitope of canine P- selectin) had no effect on the number of rolling leukocytes or on their rolling velocity. These results show for the first time that P-selectin plays an essential role in leukocyte rolling in vivo, and therefore may be a key participant of the inflammatory response.     

P-selectin glycoprotein ligand-1 is broadly expressed in cells of myeloid, lymphoid, and dendritic lineage and in some nonhematopoietic cells

P-selectin glycoprotein ligand-1 (PSGL-1) is a muclin-like glycoportein ligand for P- and E-selectin on myeloid cells and a subset of lymphocytes. We used flow cytometry and immunohistochemistry to examine expression of PSGL-1 on minor leukocyte populations, differentiating hematopoletic cells, and nonhematopoietic tissues using two monoclonal antibodies to distinct protein epitopes on PSGL-1. In the bone marrow, PSGL-1 was expressed on myeloid cells from the myeloblast stage to the segmented neutrophil, but was not detected on erythroblasts or megakaryocytes. All types of circulating myeloid cells expressed PSGL- 1, and PSGL-1 was retained after extravasation of myetoid cells into tissues. PSGL-1 was also expressed on circulating dendritic cells, monocyte-derived dendritic cells, dendritic cells in lymphoid tissues and epidermis, and follicular dendritic cells. All types of lymphoid cells examined expressed PSGK-1, including immature and mature thymocytes, naive and memory T cells, gamma/delta T cells, netural killer cells, B cells and CD34+ progenitor cells. However, PSGL-1 levels were substantially lower on tonsillar lymphocytes than on circulating lymphocytes, suggesting that PSGL-1 expression is down regulated during or after entry of lymphocytes into secondary lymphoid tissue. Although PSGL-1 antigen was detected primarily on hamatopoietic cells, it was also present on time epithelium of the fallopian tube. Furthermore, PSGL-1 antigen gen was detected sporadically on microvascular endothelium in some pathologic tissues. This suggests that PSGL-1 may have functions other than mediating leukocyte adhersion.     

Polymorphisms of P-selectin glycoprotein ligand-1 are associated with neutrophil-platelet adhesion and with ischaemic cerebrovascular disease

P-selectin glycoprotein ligand (PSGL-1) shares common features with platelet glycoprotein Ibalpha. A recently described polymorphism in this receptor that results in a variable number of tandem repeats (VNTR) sequence present either 16, 15 or 14 times (alleles A, B or C) could, similar to GPIbalpha, be functionally relevant. The allelic frequency of this polymorphism was investigated in 469 individuals from the south of Spain, and was similar to that previously described in other Caucasian populations: 85% A, 14% B and 1% C alleles. We identified two new polymorphisms genetically linked to the C isoform, resulting in the Ser273Phe and Met274Val changes. To assess the functional consequence of the polymorphisms in the receptor, we performed flow cytometric analysis of P-selectin dependent neutrophil-platelet interaction. Neutrophils carrying the shortest C allele and the amino acid variations in residues 273 and 274 exhibited a significantly lower capacity to bind activated platelets than A/B and A/A samples (mean fluorescence intensity of CD42b+ neutrophils 262 versus 303 and 319 respectively, P < 0.05). The distribution of the VNTR was analysed in three case-control studies including 104 cerebrovascular (CVD), 101 coronary heart disease (CHD) and 150 deep venous thrombosis (DVT) patients. The results showed that smaller (B and C) alleles seem to be associated with a lower risk of developing CVD (P = 0.008) but not to be related to CHD or DVT. In conclusion, polymorphisms of the PSGL-1 receptor may influence the neutrophil-platelet binding, and represent a risk factor for CVD.     

P-selectin glycoprotein ligand-1 deficiency leads to cytokine resistance and protection against atherosclerosis in apolipoprotein E deficient mice

Adhesive interactions between endothelial cells and leukocytes contribute to atherosclerotic plaque growth. However, mechanism(s) responsible for endothelial priming and deactivation in inflammatory diseases such as atherosclerosis are not clear. Apolipoprotein E deficient mice were generated with deficiency of P-selectin glycoprotein ligand-1 (Psgl-1(-/-), ApoE(-/-)). On both standard chow and Western diet, Psgl-1(-/-), ApoE(-/-) mice were protected against atherosclerosis compared to Psgl-1(+/+), ApoE(-/-) controls. Psgl-1(-/-), ApoE(-/-) mice also showed reduced leukocyte rolling and firm attachment on endothelial cells, however, adoptively transferred Psgl-1(+/+), ApoE(-/-) leukocytes into Psgl-1(-/-), ApoE(-/-) hosts displayed similar reduced rolling as Psgl-1(-/-), ApoE(-/-) leukocytes. Hematopoietic deficiency of Psgl-1 conferred resistance to the effects of interleukin-1β (IL-1β) on leukocyte rolling along with reduced circulating levels of sP-sel and sE-sel. Antibody blockade of Psgl-1 also reduced endothelial activation in response to IL-1β, eliminated leukocyte rolling, and was protective against atherosclerosis in ApoE(-/-) mice. Monocyte depletion with clodronate restored the endothelial response to IL-1β in Psgl-1(-/-) mice. This study suggests that Psgl-1 deficiency leads to reduced atherosclerosis and adhesive interactions between endothelial cells and leukocytes by indirectly regulating endothelial responses to cytokine stimulation.