P-selectin Glycoprotein Ligand-1 Interactions Research Articles

Unraveling the Mechanisms of P-Selectin in Sickle Cell Disease Using a Microvascular-on-a-Chip Model

Individuals who suffer from Sickle Cell Disease (SCD) is known to have multifaceted complications one such being vaso-occlusive crises, which result from blockages in their blood vessels. Recently, progress has been made to get drugs to the marketplace that address this issue. Several of these drugs target p-selectin, a transmembrane protein that facilitates the adhesion of platelets, leukocytes, and activated endothelial cells. P-selectin Glycoprotein Ligand-1 (PSGL-1), a P-selectin antagonist, has been extensively researched in vitro for its ability to mediate Microvascular obstruction through leukocyte interactions, particularly leukocyte rolling. Particularly, in human patients rather than animal models, the precise role of P-selectin in the interactions between activated platelets and endothelial cells remains unclear. This study delves into this interaction using a 3D vein-on-a-chip model that can capture microvascular obstruction formation at a similar scale to the microvascular. Our data shows that P Selectin in SCD inhibition is the attenuation mainly driven by platelets with contributions from WBC rather than driven by the activated endothelium. Through the creation of a PDMS branching 32-channel microfluidic, the cell interactions and obstruction of the microvascular can be quantified. Additionally, this model serves as an analog to test the various intercellular PSGL-1 interactions. Endothelial cells are seeded into the channels and cultured under flow to create 3D structures. Once confluent, healthy, and SCD patient blood samples collected in sodium citrate can be recalcified and perfused into the endothelial network to simulate physiological conditions then endothelial activation markers like P Selectin can be tagged, imaged, and quantified. Microvascular obstruction can be imaged in live time and quantified primarily through area and accumulation. Finally, various PSGL-1 analogs can be doped into the blood before and after perfusion to simulate prophylaxis or treatment. This system allows for the study of PSGL-1's effect on the microvascular obstruction process and its effect on individual cell types through tagging [Figure 1A]. When investigating PSGL1 analogs, the system does indicate a significant reduction in microvascular obstruction, quantified by the amount of occlusion within the channels [Figure 1B & 1C]. With a cohort of n=10 SS patients on Hydroxyurea, there is a downstream effect on the amount of occlusion, specifically the percentage of the area of the channel that is occluded. Each patient is compared against itself to serve as its control, and each patient is perfused into three near-identical chips of treated and three untreated [Figure 1D]. When targeting CD41 and CD45 within the model, occlusions seem to be primarily driven by activated platelets, an indication that PSGL-1's interactions may be more involved in platelets than leukocyte dynamics. Work is ongoing to target this specific interaction. PSGL1's interaction with the activated endothelium is driving microvascular obstruction to be dominated by activated platelet leukocyte interactions. Current data shows that attenuation of P Selectin decreases vascular obstruction. PSGL-1 cell interactions and PSGL-1's effect on occlusion mitigation are quantified. By quantifying PSGL-1's interactions through a vein-on-a-chip model, SCD patient-specific drug interactions can be identified. Additionally, a greater understanding of how these drugs affect vaso-occlusive crisis and SCD.

Targeted nanotherapy with everolimus reduces inflammation and fibrosis in scleroderma-related interstitial lung disease developed by PSGL-1 deficient mice.

Interstitial lung disease (ILD) is the main cause of mortality in systemic sclerosis (SSc), and current therapies available are of low efficacy or high toxicity. Thus, the identification of innovative less toxic and high efficacy therapeutic approaches to ILD treatment is an urgent need. The interaction of P-selectin glycoprotein ligand-1 (PSGL-1) with P-selectin initiates leukocyte extravasation and deletion of the corresponding gene (Selplg) induces a SSc-like syndrome with high incidence of ILD in aged mice. Aged PSGL-1 KO (Selplg-/- ) mice were used to assess the therapeutic effects of nanotherapy with everolimus, included in liposomes decorated with high MW hyaluronic acid (LipHA+Ev) and administered intratracheally to specifically target CD44-expressing lung cells. PSGL-1 KO mice had increased numbers of CD45+ and CD45- cells, including alveolar and interstitial macrophages, eosinophils, granulocytes and NK cells, and myofibroblasts in bronchoalveolar lavage (BAL). CD45+ and CD45- cells expressing pro-inflammatory and pro-fibrotic cytokines were also increased. Lungs from PSGL-1 KO mice showed increased immune cell infiltration and apoptosis and exacerbated interstitial and peribronchial fibrosis. Targeted nanotherapy with LipHA+Ev decreased the myofibroblasts in BAL, cells producing proinflammatory and profibrotic cytokines, and the degree of lung inflammation at histology. LipHA+Ev treatment also decreased the severity of peribronchial and interstitial lung fibrosis, from moderate to mild levels. In PSGL-1 KO mice, targeted nanotherapy with LipHA+Ev was an effective treatment for SSc-ILD, reducing the number of inflammatory and fibrotic cells in BAL and reducing inflammation and fibrosis in lungs.

Recombinant Human Vimentin Binds to P-Selectin and Blocks Neutrophil Capture and Rolling on Platelets and Endothelium

Leukocyte adhesion to vascular endothelium and platelets is an early step in the acute inflammatory response. The initial process is mediated through P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes binding to platelets adhered to endothelium and the endothelium itself via P-selectin. Although these interactions are generally beneficial, pathologic inflammation may occur in undesirable circumstances, such as in acute lung injury (ALI) and ischemia and reperfusion injury. Therefore, the development of novel therapies to attenuate inflammation may be beneficial. In this article, we describe the potential benefit of using a recombinant human vimentin (rhVim) on reducing human leukocyte adhesion to vascular endothelium and platelets under shear stress. The addition of rhVim to whole blood and isolated neutrophils decreased leukocyte adhesion to endothelial and platelet monolayers. Furthermore, rhVim blocked neutrophil adhesion to P-selectin-coated surfaces. Binding assays showed that rhVim binds specifically to P-selectin and not to its counterreceptor, PSGL-1. Finally, in an endotoxin model of ALI in C57BL/6J mice, treatment with rhVim significantly decreased histologic findings of ALI. These data suggest a potential role for rhVim in attenuating inflammation through blocking P-selectin-PSGL-1 interactions.

The Interaction of Selectins and PSGL-1 as a Key Component in Thrombus Formation and Cancer Progression.

Cellular interaction is inevitable in the pathomechanism of human disease. Formation of heterotypic cellular aggregates, between distinct cells of hematopoietic and nonhematopoietic origin, may be involved in events leading to inflammation and the complex process of cancer progression. Among adhesion receptors, the family of selectins with their ligands have been considered as one of the major contributors to cell-cell interactions. Consequently, the inhibition of the interplay between selectins and their ligands may have potential therapeutic benefits. In this review, we focus on the current evidence on the selectins as crucial modulators of inflammatory, thrombotic, and malignant disorders. Knowing that there is promiscuity in selectin binding, we outline the importance of a key protein that serves as a ligand for all selectins. This dimeric mucin, the P-selectin glycoprotein ligand 1 (PSGL-1), has emerged as a major player in inflammation, thrombus, and cancer development. We discuss the interaction of PSGL-1 with various selectins in physiological and pathological processes with particular emphasis on mechanisms that lead to severe disease.

Ezrin-Radixin-Moesin-binding Sequence of PSGL-1 Glycoprotein Regulates Leukocyte Rolling on Selectins and Activation of Extracellular Signal-regulated Kinases

P-selectin glycoprotein ligand-1 (PSGL-1) mediates the capture (tethering) of free-flowing leukocytes and subsequent rolling on selectins. PSGL-1 interactions with endothelial selectins activate Src kinases and spleen tyrosine kinase (Syk), leading to α(L)β(2) integrin-dependent leukocyte slow rolling, which promotes leukocyte recruitment into tissues. In addition, but through a distinct pathway, PSGL-1 engagement activates ERK. Because ezrin, radixin and moesin proteins (ERMs) link PSGL-1 to actin cytoskeleton and because they serve as adaptor molecules between PSGL-1 and Syk, we examined the role of PSGL-1 ERM-binding sequence (EBS) on cell capture, rolling, and signaling through Syk and MAPK pathways. We carried out mutational analysis and observed that deletion of EBS severely reduced 32D leukocyte tethering and rolling on L-, P-, and E-selectin and slightly increased rolling velocity. Alanine substitution of Arg-337 and Lys-338 showed that these residues play a key role in supporting leukocyte tethering and rolling on selectins. Importantly, EBS deletion or Arg-337 and Lys-338 mutations abrogated PSGL-1-induced ERK activation, whereas they did not prevent Syk phosphorylation or E-selectin-induced leukocyte slow rolling. These studies demonstrate that PSGL-1 EBS plays a critical role in recruiting leukocytes on selectins and in activating the MAPK pathway, whereas it is dispensable to phosphorylate Syk and to lead to α(L)β(2)-dependent leukocyte slow rolling.

P-selectin Glycoprotein Ligand-1 Decameric Repeats Regulate Selectin-dependent Rolling under Flow Conditions

P-selectin glycoprotein ligand-1 (PSGL-1) interacts with selectins to support leukocyte rolling along vascular wall. L- and P-selectin bind to N-terminal tyrosine sulfate residues and to core-2 O-glycans attached to Thr-57, whereas tyrosine sulfation is not required for E-selectin binding. PSGL-1 extracellular domain contains decameric repeats, which extend L- and P-selectin binding sites far above the plasma membrane. We hypothesized that decamers may play a role in regulating PSGL-1 interactions with selectins. Chinese hamster ovary cells expressing wild-type PSGL-1 or PSGL-1 molecules exhibiting deletion or substitution of decamers with the tandem repeats of platelet glycoprotein Ibalpha were compared in their ability to roll on selectins and to bind soluble L- or P-selectin. Deletion of decamers abrogated soluble L-selectin binding and cell rolling on L-selectin, whereas their substitution partially reversed these diminutions. P-selectin-dependent interactions with PSGL-1 were less affected by decamer deletion. Videomicroscopy analysis showed that decamers are required to stabilize L-selectin-dependent rolling. Importantly, adhesion assays performed on recombinant decamers demonstrated that they directly bind to E-selectin and promote slow rolling. Our results indicate that the role of decamers is to extend PSGL-1 N terminus far above the cell surface to support and stabilize leukocyte rolling on L- or P-selectin. In addition, they function as a cell adhesion receptor, which supports approximately 80% of E-selectin-dependent rolling.

P-selectin glycoprotein ligand-1 mediates L-selectin-independent leukocyte rolling in high endothelial venules of peripheral lymph nodes

Lymphocyte homing to peripheral lymph nodes (LNs) requires L-selectin. Previous studies, however, suggest that there are L-selectin-independent mechanisms of lymphocyte homing. P-selectin glycoprotein ligand-1 (PSGL-1) is a major ligand for P-selectin expressed in a selectin-binding form on myeloid cells and subsets of lymphoid cells. To discover whether PSGL-1 plays a role in lymphocyte homing, we examined leukocyte rolling and adhesion in the high endothelial venules (HEVs) of the subiliac LNs of wild-type and PSGL-1-deficient mice by intravital microscopy. There were no significant differences in blood velocity or wall shear stress between wild-type and PSGL-1-deficient mice. Although the leukocyte rolling fraction was not altered in PSGL-1-deficient mice, infusion of an anti-L-selectin mAb into these mice completely abolished leukocyte rolling, while the same treatment in wild-type mice inhibited 90% of the leukocyte rolling. This residual rolling in wild-type mice appears to depend on the PSGL-1-P-selectin interaction, since infusion of an anti-L-selectin mAb together with an anti-PSGL-1 mAb or anti-P-selectin mAb almost completely abolished the rolling. PSGL-1 deficiency also led to a higher rolling velocity, suggesting that PSGL-1 mediates leukocyte rolling at low velocities. P-selectin was found to be expressed on the HEVs of subiliac LNs under the conditions of intravital microscopy. Taken together, these results indicate that the interaction of PSGL-1 with P-selectin constitutes a second mechanism of leukocyte rolling in the HEVs of peripheral LNs.

Biomechanics of P-Selectin PSGL-1 Bonds: Shear Threshold and Integrin-Independent Cell Adhesion

Platelet-leukocyte adhesion may contribute to thrombosis and inflammation. We examined the heterotypic interaction between unactivated neutrophils and either thrombin receptor activating peptide (TRAP)-stimulated platelets or P-selectin-bearing beads (Ps-beads) in suspension. Cone-plate viscometers were used to apply controlled shear rates from 14 to 3000/s. Platelet-neutrophil and bead-neutrophil adhesion analysis was performed using both flow cytometry and high-speed videomicroscopy. We observed that although blocking antibodies against either P-selectin or P-selectin glycoprotein ligand-1 (PSGL-1) alone inhibited platelet-neutrophil adhesion by ∼60% at 140/s, these reagents completely blocked adhesion at 3000/s. Anti-Mac-1 alone did not alter platelet-neutrophil adhesion rates at any shear rate, though in synergy with selectin antagonists it abrogated cell binding. Unstimulated neutrophils avidly bound Ps-beads and activated platelets in an integrin-independent manner, suggesting that purely selectin-dependent cell adhesion is possible. In support of this, antagonists against P-selectin or PSGL-1 caused dissociation of previously formed platelet-neutrophil and Ps-bead neutrophil aggregates under shear in a variety of experimental systems, including in assays performed with whole blood. In studies where medium viscosity and shear rate were varied, a shear threshold for P-selectin PSGL-1 binding was also noted at shear rates <100/s when Ps-beads collided with isolated neutrophils. Results are discussed in light of biophysical computations that characterize the collision between unequal-size particles in linear shear flow. Overall, our studies reveal an integrin-independent regime for cell adhesion and weak shear threshold for P-selectin PSGL-1 interactions that may be physiologically relevant.

Quantifying the effects of contact duration, loading rate, and approach velocity on P-selectin–PSGL-1 interactions using AFM

Kinetics and its regulation by extrinsic physical factors govern selectin–ligand interactions that mediate tethering and rolling of circulating cells on the vessel wall under hemodynamic forces. While the force regulation of off-rate for dissociation of selectin–ligand bonds has been extensively studied, much less is known about how transport impacts the on-rate for association of these bonds and their stability. We used atomic force microscopy (AFM) to quantify how the contact duration, loading rate, and approach velocity affected kinetic rates and strength of bonds of P-selectin interacting with P-selectin glycoprotein ligand 1 (PSGL-1). We found a saturable relationship between the contact time and the rupture force, a biphasic relationship between the adhesion probability and the retraction velocity, a piece-wise linear relationship between the rupture force and the logarithm of the loading rate, and a threshold relationship between the approach velocity and the rupture force. These results provide new insights into how physical factors regulate receptor–ligand interactions.

Regulation of PSGL-1 Interactions with L-selectin, P-selectin, and E-selectin: ROLE OF HUMAN FUCOSYLTRANSFERASE-IV AND -VII

P-selectin glycoprotein ligand-1 (PSGL-1) interactions with selectins regulate leukocyte migration in inflammatory lesions. In mice, selectin ligand activity regulating leukocyte recruitment and lymphocyte homing into lymph nodes results from the sum of unequal contributions of fucosyltransferase (FucT)-IV and FucT-VII, with FucT-VII playing a predominant role. Here we have examined the role of human FucT-IV and -VII in conferring L-selectin, P-selectin, and E-selectin binding activities to PSGL-1. Lewis x (Le(x)) carbohydrate was generated at the CHO(dhfr)(-) cell surface by FucT-IV expression, whereas sialyl Le(x) (sLe(x)) was synthesized by FucT-VII. Both human FucT-IV and -VII had the ability to generate carbohydrate ligands that support L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1, with FucT-VII playing a major role. Cooperation was observed between FucT-IV and -VII in recruiting L-, P-, or E-selectin-expressing cells on PSGL-1 and in regulating cell rolling velocity and stability. Additional rolling adhesion assays were performed to assess the role of Thr-57-linked core-2 O-glycans in supporting L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1. These studies confirmed that core-2 O-glycans attached to Thr-57 play a critical role in supporting L- and P-selectin-dependent rolling and revealed that additional binding sites support >75% of E-selectin-mediated rolling. The observations presented here indicate that human FucT-IV and -VII both contribute and cooperate in regulating L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1, with FucT-VII playing a predominant role in conferring selectin binding activity to PSGL-1.

Increased Platelet Binding to Circulating Monocytes in Acute Coronary Syndromes

Present therapies for acute coronary syndromes aim toward limiting platelet-platelet adhesion and aggregation processes. However, platelet-leukocyte interactions may contribute importantly to disease progression in the arterial wall. Recent studies suggest that prevention of platelet-leukocyte binding via P-selectin glycoprotein ligand-1 (PSGL-1) may be beneficial in animal models of vascular injury. P-selectin-PSGL-1 interactions were found to account for most platelet-monocyte binding observed in peripheral blood samples from healthy donors. However, a significant component of observed adhesion was calcium independent, involving neither PSGL-1 nor P-selectin. Platelet-monocyte interactions were examined in 52 patients admitted within 14 hours of symptom onset, with acute coronary syndromes defined as unstable angina (n=12) and acute myocardial infarction (n=13) or noncardiac chest pain (n=27). When compared with patients with noncardiac chest pain, significantly elevated levels of platelet-monocyte binding were found in patients with acute myocardial infarction (70.1+/-15.4% versus 45.4+/-23.3%; P<0.01) and unstable angina (67.4+/-12.9% versus 45.4+/-23.3%; P>0.01). Calcium-independent platelet-monocyte binding was significantly elevated in myocardial infarction patients alone (14.7+/-7.7% versus 6.1+/-5.96%; P<0.001). There is evidence for a significant P-selectin-independent molecular component to the platelet-monocyte conjugation observed in peripheral blood. Patients with myocardial infarction and unstable angina demonstrate increased total binding of platelets to monocytes. Additionally, calcium-independent adhesion was significantly elevated in patients with evidence of myocardial infarction. These findings demonstrate that novel cation-independent adhesion mechanisms may mediate platelet-monocyte binding, representing a new therapeutic target after vascular injury associated with myocardial infarction.

Selectin-Mediated Interactions Regulate Cytokine Networks and Macrophage Heme Oxygenase-1 Induction in Cardiac Allograft Recipients

Host sensitization to major histocompatibility complex (MHC) antigens is among the most critical of problems facing heart transplantation. Selectins are postulated to mediate the early adhesive events in the recruitment of leukocytes at the allograft site. We investigated the significance of selectin–P-selectin glycoprotein ligand-1 (PSGL-1)–mediated in vivo interactions in the immune cascade leading to rejection of cardiac allografts in skin presensitized rats. Infusion of a soluble recombinant form of PSGL-1 (rPSGL-Ig) during skin graft–mediated sensitization prevented Day 1.0 ± 0.1 “accelerated” rejection in sensitized rat recipients, and prolonged cardiac allograft survival to Day 3.8 ± 1.0 (p < 0.001). This therapy significantly depressed serum IgM levels and decreased intragraft expression of Th1 type cytokines (IL-2 and IFN-γ) as well as of IL-1β and MCP-1, as compared with controls, without affecting the initial number of infiltrating mononuclear cells (MNC). A profound decrease in graft-infiltrating MNC was recorded at 24 hours in rPSGL-Ig–treated rats. The expression of heme oxygenase-1 (HO-1), an inducible heat shock protein 32 that protects against oxidative cell/tissue injury, was found in approximately 14-fold higher levels in the rPSGL-Ig–treated recipients as compared with controls. The HO-1 overexpression in rPSGL-Ig–treated hosts, primarily by infiltrating macrophages, was accompanied by virtual absence of myocardial infarcts and decreased frequency of TUNEL + cells at the graft site. Moreover, down-regulation of HO-1 expression by zinc protoporphyrin, an HO-1 antagonist, decreased expression of antiapoptotic Bag-1 molecule in recipients conditioned with rPSGL-Ig. Thus, the blockade of selectin–PSGL-1 interactions depresses intracardiac allograft expression of Th1 type cytokines, and might inhibit the differentiation of Th1 type cells. In addition, it up-regulates HO-1 expression and protects against myocardial infarction and apoptosis. Hence, this study reports on a previously unrecognized role of selectin–PSGL-1–mediated interactions after in vivo alloantigenic challenge.

Polarization and interaction of adhesion molecules P-selectin glycoprotein ligand 1 and intercellular adhesion molecule 3 with moesin and ezrin in myeloid cells

In response to the chemoattractants interleukin 8, C5a,N-formyl-methionyl-leucyl-phenylalanine, and interleukin 15, adhesion molecules P-selectin glycoprotein ligand 1 (PSGL-1), intercellular adhesion molecule 3 (ICAM-3), CD43, and CD44 are redistributed to a newly formed uropod in human neutrophils. The adhesion molecules PSGL-1 and ICAM-3 were found to colocalize with the cytoskeletal protein moesin in the uropod of stimulated neutrophils. Interaction of PSGL-1 with moesin was shown in HL-60 cell lysates by isolating a complex with glutathione S-transferase fusions of the cytoplasmic domain of PSGL-1. Bands of 78- and 81-kd were identified as moesin and ezrin by Western blot analysis. ICAM-3 and moesin also coeluted from neutrophil lysates with an anti-ICAM-3 immunoaffinity assay. Direct interaction of the cytoplasmic domains of ICAM-3 and PSGL-1 with the amino-terminal domain of recombinant moesin was demonstrated by protein-protein binding assays. These results suggest that the redistribution of PSGL-1 and its association with intracellular molecules, including the ezrin-radixin-moesin actin-binding proteins, regulate functions mediated by PSGL-1 in leukocytes stimulated by chemoattractants.

Polarization and interaction of adhesion molecules P-selectin glycoprotein ligand 1 and intercellular adhesion molecule 3 with moesin and ezrin in myeloid cells

AbstractIn response to the chemoattractants interleukin 8, C5a,N-formyl-methionyl-leucyl-phenylalanine, and interleukin 15, adhesion molecules P-selectin glycoprotein ligand 1 (PSGL-1), intercellular adhesion molecule 3 (ICAM-3), CD43, and CD44 are redistributed to a newly formed uropod in human neutrophils. The adhesion molecules PSGL-1 and ICAM-3 were found to colocalize with the cytoskeletal protein moesin in the uropod of stimulated neutrophils. Interaction of PSGL-1 with moesin was shown in HL-60 cell lysates by isolating a complex with glutathione S-transferase fusions of the cytoplasmic domain of PSGL-1. Bands of 78- and 81-kd were identified as moesin and ezrin by Western blot analysis. ICAM-3 and moesin also coeluted from neutrophil lysates with an anti-ICAM-3 immunoaffinity assay. Direct interaction of the cytoplasmic domains of ICAM-3 and PSGL-1 with the amino-terminal domain of recombinant moesin was demonstrated by protein-protein binding assays. These results suggest that the redistribution of PSGL-1 and its association with intracellular molecules, including the ezrin-radixin-moesin actin-binding proteins, regulate functions mediated by PSGL-1 in leukocytes stimulated by chemoattractants.

PSGL-1Ig, A P-SELECTIN ANTAGONIST, PROLONGS CARDIAC ALLOGRAFT SURVIVAL AND ARRESTS MONONUCLEAR CELL TRAFFIC IN PRESENSITIZED RAT RECIPIENTS

217 Leukocyte recruitment at the graft (Tx) site is regulated by a sequence of adhesive and signaling events. The interactions between P/E- selectins and P-selectin glycoprotein ligand-1 (PSGL-1) mediate the earliest events in the adhesion cascade by promoting the tethering and rolling of leukocytes on the luminal surface of endothelial cells. P-selectin (CD62), a selectin preferentially expressed by endothelial cells, is one of the earliest adhesion molecules expressed locally at the Tx site during the rejection cascade. This study was designed to evaluate the significance of selectin-mediated in vivo interactions in a well-defined model of accelerated rejection of cardiac Tx in presensitized rats. LEW rats were challenged with skin Tx from BN donors (day −7), followed 1 week later by transplantation of LBNF1 hearts (day 0). These cardiac Tx were rejected in an accelerated manner in <24 h (n=5). PSGL-1Ig, a recombinant soluble form of the P-selectin glycoprotein ligand-1, was administered (100 μg/day/rat, i.v.) according to three distinct protocols. Group 1 received PSGL-1Ig between day 0 and day +2 post-Tx, Group 2 between day −7 and −1, whereas Group 3 between day −7 and day +2. Control recipients were given PBS only. In Gr. 1, treatment with PSGL-1Ig failed to affect <24 h rejection of cardiac Tx in sensitized rats (n=5). In contrast, cardiac Tx survival in Gr. 2 recipients that received a course of PSGL-1 during the sensitization phase (day −7 to −1) was significantly increased to 3.4±1.0 days, as compared to controls (1.0±0.3; p<0.001, n=6/group). Extended pre- and post-Tx treatment with PSGL-1Ig in Gr. 3 did not improve further Tx survival (3±1 days, n=3). In an attempt to dissect putative mechanisms of PSGL-1Ig-facilitated in vivo effects, cardiac Tx were harvested at 24 h from Gr. 2 recipients, and analyzed by immunohistochemistry and competitive RT-PCR. PSGL-1Ig blocked but did not depress endothelial CD62 expression. Strikingly, this therapy profoundly decreased Tx infiltration with mononuclear cells (MNC), as compared to controls (cells/10 HPF; n=3-4 /group); T cells: 18±10 vs. 67±3 (p<0.001), OX8+ lymphocytes: 34±18 vs. >200 (p<0.0001), monocytes/macrophages: 57±32 vs. >200 (p<0.001). In addition, PSGL-1Ig treatment diminished intra-Tx cellular activation, as evidenced by staining for IL-2R+ cells (14±7 vs. 43±6, p<0.01) or RT-PCR-based IL-2 mRNA expression (1.9×103 vs. 6.6×104 transcripts/μg RNA). The expression of RANTES, a monocyte chemoattractant, was also depressed in PSGL-1Ig-pretreated cardiac Tx. The fact that PSGL-1Ig-mediated therapeutic effects were relatively short-lived, and that post-Tx continuous treatment did not improve Tx survival, may imply the role of other adhesion molecules in the cascade of events that restored Tx rejection in our studies. Indeed, PSGL-1Ig treatment did not affect endothelial expression of adhesion molecules such as VCAM-1, ICAM-1, and cellular fibronectin. This is the first report that documents the in vivo role of CD62 - PSGL-1 interactions in MNC traffic and infiltration in Tx recipients. As blockage of these interactions inhibits very early intra-Tx MNC deposition, with resultant prolongation of Tx survival, PSGL-1Ig may represent a novel immunomodulatory agent for adjunctive therapy in organ transplantation.

Isolated P-selectin glycoprotein ligand-1 dynamic adhesion to P- and E-selectin.

Leukocyte adhesion to vascular endothelium under flow involves an adhesion cascade consisting of multiple receptor pairs that may function in an overlapping fashion. P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin have been implicated in neutrophil adhesion to P- and E-selectin under flow conditions. To study, in isolation, the interaction of PSGL-1 with P- and E-selectin under flow, we developed an in vitro model in which various recombinant regions of extracellular PSGL-1 were coupled to 10-microm-diameter microspheres. In a parallel plate chamber with well defined flow conditions, live time video microscopy analyses revealed that microspheres coated with PSGL-1 attached and rolled on 4-h tumor necrosis factor-alpha-activated endothelial cell monolayers, which express high levels of E-selectin, and CHO monolayers stably expressing E- or P-selectin. Further studies using CHO-E and -P monolayers demonstrate that the first 19 amino acids of PSGL-1 are sufficient for attachment and rolling on both E- and P-selectin and suggest that a sialyl Lewis x-containing glycan at Threonine-16 is critical for this sequence of amino acids to mediate attachment to E- and P-selectin. The data also demonstrate that a sulfated, anionic polypeptide segment within the amino terminus of PSGL-1 is necessary for PSGL-1-mediated attachment to P- but not to E-selectin. In addition, the results suggest that PSGL-1 has more than one binding site for E-selectin: one site located within the first 19 amino acids of PSGL-1 and one or more sites located between amino acids 19 through 148.