P-selectin Glycoprotein Ligand-1 Deficiency Research Articles

PSGL-1 deficiency re-wires T cell signaling to support development of stem cell like exhausted T cells that control PD-1 blockade resistant melanoma tumor growth

Abstract We previously identified that the adhesion molecule P-selectin glycoprotein ligand-1 (PSGL-1) regulates CD8 +T cell effector functions and promotes exhaustion in responses to chronic viral infection and melanoma tumors. In this study, we investigated cellular and molecular mechanisms by which intrinsic PSGL-1 signaling regulates the differentiation of exhausted T cells (T EX). We show that PSGL-1 localizes with the TCR upon co-ligation and find that PSGL-1 associates with Zap70 and the Zap70 inhibitor, Sts-1. In the absence of PSGL-1, expression of Sts-1 is reduced allowing for greater activation of Zap70 and the downstream signaling molecules Akt and Erk1/2. In addition, PSGL-1-deficiency empowers CD8 +T cells to respond to low affinity TCR ligands. In vivo, PSGL-1 deficiency leads to growth inhibition of a PD-1-blockade resistant melanoma. Notably, PSGL-1-deficiency in T cells enables retention of effector T cell (T EFF) functions, and an increased representation of stem cell-like T cells (T SC) that express TCF-1 and low levels of TOX, which are associated with sustained effector responses and responsiveness to ICB. We now show that concomitant PSGL-1 signaling with repeated TCR stimulation of human T EFFelicits functional exhaustion. Importantly, blockade of PSGL-1 recapitulates PSGL-1 deficiency in anti-tumor as well as antiviral responses, and its distinct mechanism of T cell inhibition underscores the translational potential of targeting PSGL-1 by ICB. Supported by grants from NIH (R01 AI106895, R21 CA249353, R21 CA216678, R03 CA252144) Melanoma Research Alliance MRA 696326, Department of Defense W81XWH-20-1-0324, Sanford Burnham Prebys NCI-Designated Cancer Center Support Grant, P30 CA030199 American Cancer Society Postdoctoral Fellowship (PF-20-113-01-LIB)

P-Selectin Glycoprotein Ligand-1 Deficiency Protects Against Aortic Aneurysm Formation Induced by DOCA Plus Salt.

P-selectin glycoprotein ligand-1 (PSGL-1) acts as a crucial regulator for the inflammatory cells infiltration by mediating the adhesion of leukocytes. However, the role of PSGL-1 in aortic aneurysm remains elusive. Here, we investigated the role of PSGL-1 in aortic aneurysm (AA) development. We first detected PSGL-1 expression in samples from aortic aneurysm patients and mouse AA models via western blotting, immunofluorescence, and flow cytometry, and then we used global PSGL-1 knockout mice and their wild type controls to establish an aortic aneurysm model induced by deoxycorticosterone acetate (DOCA) plus high salt (HS). The incidence, fatality rates, and the pathological changes of aortic aneurysm were analyzed in each group. The inflammation, adhesion molecules expression, and PSGL-1 mediated leukocyte-endothelial adhesion and their underlying mechanisms were explored further. Increased PSGL-1 levels were observed in human and mouse aortic aneurysm, and on leukocytes of mice treated with DOCA+HS. PSGL-1 deficiency reduced the incidence and severity of aortic aneurysm significantly, as well as decreased elastin fragmentation, collagen accumulation, and smooth muscle cells degeneration. Mechanistically, the protective effect of PSGL-1 inhibition was mediated by the reduced adhesion molecules, and the subsequently reduced leukocyte-endothelial adhesion through the NF-κB pathway, which finally led to reduced inflammatory cells infiltration and decreased inflammatory factors expression. PSGL-1 deficiency is protective against inflammatory cells migration and recruitment in the condition of AA through attenuation of leukocyte-endothelial adhesion. Inhibition of PSGL-1 may be a potential therapeutic target for the prevention and treatment of human AA.

P-selectin glycoprotein ligand 1 deficiency prevents development of acute pancreatitis by attenuating leukocyte infiltration.

BACKGROUNDAcute pancreatitis (AP) is rapid-onset pancreatic inflammation that causes local and systemic inflammatory response syndrome (SIRS) with high morbidity and mortality, but no approved therapies are currently available. P-selectin glycoprotein ligand 1 (PSGL-1) is a transmembrane glycoprotein to initiate inflammatory responses. We hypothesized that PSGL-1 may be involved in the development of AP and would be a new target for the treatment of AP.AIMTo investigate the role and mechanism of PSGL-1 in the development of AP.METHODSThe PSGL-1 expression on leukocytes was detected in peripheral blood of AP patients and volunteers. Pancreatic injury, inflammatory cytokines expression, and inflammatory cell infiltration was measured in AP mouse models induced with PSGL-1 knockout (PSGL-1-/-) and wild-type (PSGL-1+/+) mice. Leukocyte-endothelial cell adhesion was measured in a peripheral blood mononuclear cell (PBMC)-endothelial cell coculture system.RESULTSThe expression of PSGL-1 on monocytes and neutrophils was significantly increased in AP patients. Compared with PSGL-1+/+ mice, PSGL-1-/- AP mice induced by caerulein exhibited lower serum amylase, less Interleukin-1beta (IL-1beta) and Interleukin-6 (IL-6) expression, less neutrophil and macrophage infiltration, and reduced peripheral neutrophil and monocyte accounts. PSGL-1 deficiency alleviated leukocyte-endothelial cell adhesion via IL-6 but not IL-1beta.CONCLUSIONPSGL-1 deficiency effectively inhibits the development of AP by preventing leukocyte-endothelial cell adhesion via IL-6 stimulation and may become a potential therapeutic target for treating AP.

Enhanced ER stress responses accompany greater effector T cell functions with deficiency of PSGL-1

Abstract Endoplasmic reticulum (ER) stress is induced by the accumulation of misfolded proteins and other cellular stresses, which lead to the unfolded protein response to restore ER homeostasis. Previous studies have shown that the ER stress response is required for T cell activation and differentiation. However, recent studies demonstrated that inhibition or deletion of ER stress mediators in T cells actually promotes anti-tumor immunity. Therefore, this incongruity as to the role of ER stress response in effector T cell function warrants further investigation. Our lab previously identified that the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1), acts as a T cell immune checkpoint regulator. PSGL-1-deficiency enhances T cell function, resulting in the clearance of chronic viral infection and dramatic tumor control. To better understand the contribution of ER stress to T cell function, we assessed T cell phenotype and function together with the ER stress mediators, XBP1s and ATF4, in highly-functional PSGL-1−/− T cells as well as exhausted wild-type T cells in both tumor and virus models. In melanoma models, ER stress was highly induced in activated tumor-infiltrating T cells (TILs). Effector CD8+ TILs producing IFNg displayed elevated XBP1s and ATF4 compared to IFNg− TILs. Upon chronic LCMV Cl13 viral infection in PSGL-1−/− mice, we found increased virus-specific T cells with enhanced multi-functional effector responses and increased memory/stem cell-like T cell populations characterized by the expression of the transcription factor TCF-1, as well as increased ER stress responses. Together, these data suggest that ER stress plays an essential role in effector T cell functions in settings of chronic antigen stimulation.

Angiotensin II-induced Hypertension is Reduced by Deficiency of P-selectin Glycoprotein Ligand-1

Identification of inflammatory mediators that regulate the vascular response to vasopressor molecules may aid in the development of novel therapeutic agents to treat or prevent hypertensive vascular diseases. Leukocytes have recently been shown to be capable of modifying blood pressure responses to vasopressor molecules. The purpose of this study was to test the hypothesis that deficiency of the leukocyte ligand, Psgl-1, would reduce the pressor response to angiotensin II (Ang II). Mice deficient in Psgl-1 (Psgl-1−/−) along with wild-type (WT) controls were treated for 2 weeks with a continuous infusion of Ang II. No differences in blood pressure between the groups were noted at baseline, however after 5 days of Ang II infusion, systolic blood pressures were higher in WT compared to Psgl-1−/− mice. The pressor response to acute administration of high dose Ang II was also attenuated in Psgl-1−/− compared to WT mice. Chimeric mice with hematopoietic deficiency of Psgl-1 similarly showed a reduced pressor response to Ang II. This effect was associated with reduced plasma interleukin-17 (IL-17) levels in Psgl-1−/− mice and the reduced pressor response was restored by administration of recombinant IL-17. In conclusion, hematopoietic deficiency of Psgl-1 attenuates Ang II-induced hypertension, an effect that may be mediated by reduced IL-17.

The metabonomics study of P-selectin glycoprotein ligand-1 (PSGL-1) deficiency inhibiting the progression of atherosclerosis in LDLR-/- mice.

Atherosclerosis (AS) is a multi-factorial chronic disease commonly associated with the mechanisms of metabolism disorder, endothelial dysfunction and chronic inflammation. AS an inflammatory molecule, p-selectin glycoprotein ligand-1 (PSGL-1) played an important role in the inflammatory process of atherogenesis involving the recruitment of leukocyte and transmitting signals to activate leukocyte during the adhesion process. So far, there has been little study regarding the effects of PSGL-1 on AS progression and the metabolic regulation. In this report, we studied the effect of PSGL-1 deficiency on the formation and progression of AS and the metabolic regulation by use of LDLR-/-, PSGL-1-/- transgenic mice based on metabonomics. It was found that the PSGL-1 deficiency reduced the atherosclerotic plaque area, inflammatory cells infiltration and fiber hyperplasia during the AS development. The serum metabonomics study showed that the LDLR-/- ,PSGL-1-/- mice had higher levels of HDL, valine, acetate, pyruvate, choline, PC, GPC and glycine, and lower levels of LDL+VLDL and lactate at the early stage of atherosclerosis, while lactate, citrate and glutamine showed statistical significance at the late stage of atherosclerosis. These results showed that the PSGL-1 deficiency inhibited the AS progression and regulated glucose metabolism, lipid metabolism, amino acid and phospholipid metabolism in LDLR-/- mice.

Activated signature of antiphospholipid syndrome neutrophils reveals potential therapeutic target.

Antiphospholipid antibodies, present in one-third of lupus patients, increase the risk of thrombosis. We recently reported a key role for neutrophils - neutrophil extracellular traps (NETs), in particular - in the thrombotic events that define antiphospholipid syndrome (APS). To further elucidate the role of neutrophils in APS, we performed a comprehensive transcriptome analysis of neutrophils isolated from patients with primary APS. Moreover, APS-associated venous thrombosis was modeled by treating mice with IgG prepared from APS patients, followed by partial restriction of blood flow through the inferior vena cava. In patients, APS neutrophils demonstrated a proinflammatory signature with overexpression of genes relevant to IFN signaling, cellular defense, and intercellular adhesion. For in vivo studies, we focused on P-selectin glycoprotein ligand-1 (PSGL-1), a key adhesion molecule overexpressed in APS neutrophils. The introduction of APS IgG (as compared with control IgG) markedly potentiated thrombosis in WT mice, but not PSGL-1-KOs. PSGL-1 deficiency was also associated with reduced leukocyte vessel wall adhesion and NET formation. The thrombosis phenotype was restored in PSGL-1-deficient mice by infusion of WT neutrophils, while an anti-PSGL-1 monoclonal antibody inhibited APS IgG-mediated thrombosis in WT mice. PSGL-1 represents a potential therapeutic target in APS.

PSGL-1 Is an Immune Checkpoint Regulator that Promotes T Cell Exhaustion

Chronic viruses and cancers thwart immune responses in humans by inducing Tcell dysfunction. Using a murine chronic virus that models human infections, we investigated the function of the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1), that is upregulated on responding Tcells. PSGL-1-deficient mice cleared the virus due to increased intrinsic survival of multifunctional effector Tcells that had downregulated PD-1 as well as other inhibitory receptors. Notably, this response resulted in CD4(+)-T-cell-dependent immunopathology. Mechanistically, PSGL-1 ligation on exhausted CD8(+) Tcells inhibited Tcell receptor (TCR) and interleukin-2 (IL-2) signaling and upregulated PD-1, leading to diminished survival with TCR stimulation. In models of melanoma cancer in which Tcell dysfunction occurs, PSGL-1 deficiency led to PD-1 downregulation, improved Tcell responses, and tumor control. Thus, PSGL-1 plays a fundamental role in balancing viral control and immunopathology and also functions to regulate Tcell responses in the tumor microenvironment.

PSGL-1, an immune checkpoint regulator that promotes T cell exhaustion in chronic viral infection and cancer (VIR10P.1166)

Abstract Chronic viruses, including HIV and hepatitis B and C, can survive in humans by inducing T cell dysfunction. Using a murine chronic virus that models human infections, we investigated the function of the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1) that is upregulated on responding T cells. PSGL-1-deficient mice unexpectedly cleared the virus due to dramatic increases in the intrinsic survival of multifunctional effector T cells that had downregulated PD-1 and other inhibitory receptors. Notably, this response resulted in immunopathology requiring CD4+ T cells. Mechanistically, PSGL-1 ligation on exhausted CD8+ T cells sustained PD-1 expression and diminished their survival during TCR stimulation. In a model of malignant melanoma where T cell dysfunction also occurs, PSGL-1 deficiency led to PD-1 downregulation, improved T cell responses and tumor control. Thus, PSGL-1 not only plays a fundamental role in balancing viral control and immunopathology, but also functions as a checkpoint that regulates T cell function in the tumor microenvironment.

Development of an autoimmune syndrome affecting the skin and internal organs in P-selectin glycoprotein ligand 1 leukocyte receptor-deficient mice.

To define and characterize the progression of the spontaneous autoimmune disease that develops in mice in the absence of the leukocyte adhesion receptor P-selectin glycoprotein ligand 1 (PSGL-1). Skin-resident immune cells from PSGL-1-deficient mice and C57BL/6 control mice of different ages were isolated and analyzed by flow cytometry. Biochemical parameters were analyzed in mouse serum and urine, and the presence of serum autoantibodies was investigated. Skin and internal organs were extracted, and their structure was analyzed histologically. Skin-resident innate and adaptive immune cells from PSGL-1(-/-) mice had a proinflammatory phenotype with an imbalanced T effector cell:Treg cell ratio. Sera from PSGL-1(-/-) mice had circulating autoantibodies commonly detected in connective tissue-related human autoimmune diseases. Biochemical and histologic analysis of skin and internal organs revealed skin fibrosis and structural and functional abnormalities in the lungs and kidneys. Furthermore, PSGL-1(-/-) mice exhibited vascular alterations, showing loss of dermal vessels, small vessel medial layer remodeling in the lungs and kidneys, and ischemic processes in the kidney that promote renal infarcts. Our study demonstrates that immune system overactivation due to PSGL-1 deficiency triggers an autoimmune syndrome with characteristics similar to systemic sclerosis, including skin fibrosis, vascular alterations, and systemic organ involvement. These results suggest that PSGL-1 expression contributes to the maintenance of the homeostasis of the immune system and could act as a barrier for autoimmunity in mice.

P-selectin glycoprotein ligand-1 deficiency augments G-CSF induced myeloid cell mobilization

The effect of granulocyte colony-stimulating factor (G-CSF) was investigated in P-selectin glycoprotein ligand-1 (PSGL-1) deficient (PSGL-1(-/-)) and wild-type (PSGL-1(+/+)) mice to establish the role of this mucin in myeloid cell mobilization. G-CSF activates tissue proteases that cleave adhesion molecules, thus enhances the mobilization of myeloid cells and haematopoietic stem cells. Cytopenia was induced with a single dose of cyclophosphamide. In PSGL-1(-/-) animals, we observed a delayed extravasation of mature myeloid cells from the peripheral vessels into the tissue compartments and their faster mobilization from the bone marrow. Subsequently, animals received G-CSF twice a day for 4days. Neutrophil and monocyte counts increased upon completion of G-CSF treatment and both values were significantly higher in PSGL-1(-/-) mice; 47.7 versus 28.3G/l for neutrophils and 4.1 versus 2.0G/l for monocytes. The ratio of atypical myeloid cells was also elevated. Analyzing the causes of the above differences, we identified a 4-fold increase in the colony-forming unit (CFU-GM) counts of the peripheral blood in PSGL-1(-/-) mice, compared to wild-type animals. A significantly elevated number of CFU-GM was detected also in the femurs of PSGL-1(-/-) mice, 4 and 5days after cyclophosphamide treatment and these values paralleled with the elevation of CD34+/CD117+ stem cell counts in the peripheral blood. Our data suggest, that in the absence of PSGL-1, G-CSF was more potent in elevating absolute myeloid cell numbers by acting on cell release from the bone marrow, maturation from circulating precursor cells in the peripheral blood and prolonged retainment in the circulation.

Obesity-Induced Endothelial Dysfunction Is Prevented by Deficiency of P-Selectin Glycoprotein Ligand-1

Endothelial dysfunction precedes atherosclerosis and represents an important link between obesity and cardiovascular events. Strategies designed to prevent endothelial dysfunction may therefore reduce the cardiovascular complications triggered by obesity. We tested the hypothesis that deficiency of P-selectin glycoprotein ligand-1 (Psgl-1) would improve the endothelial dysfunction associated with obesity. Psgl-1-deficient (Psgl-1−/−) and wild-type (Psgl-1+/+) mice were fed standard chow or a high-fat, high-sucrose diet (diet-induced obesity [DIO]) for 10 weeks. DIO increased mesenteric perivascular adipose tissue (mPVAT) macrophage content and vascular oxidative stress in Psgl-1+/+ mice but not in Psgl-1−/− mice. Pressure myography using mesenteric arteries demonstrated that relaxation responses to acetylcholine were significantly impaired in DIO Psgl-1+/+ mice, whereas DIO Psgl-1−/− mice were protected from endothelial dysfunction with similar relaxation responses to Psgl-1+/+ or Psgl-1−/− mice fed standard chow. The superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL) partially recovered impaired endothelial function induced by DIO. A neutralizing Psgl-1 antibody was also effective in preventing endothelial dysfunction and reducing mPVAT macrophage content induced by DIO. These results indicate that obesity in mice leads to PVAT inflammation and endothelial dysfunction that is prevented by Psgl-1 deficiency. Psgl-1 inhibition may be a useful treatment strategy for targeting vascular disease associated with obesity.

Abstract 9071: P-Selectin Glycoprotein Ligand-1 Deficiency is Protective Against Perivascular Macrophage Infiltration and Obesity-Induced Endothelial Dysfunction

Introduction: P-selectin glycoprotein ligand-1 (Psgl-1) is a leukocyte ligand which binds to selectins and mediates leukocyte trafficking in several disease states. The goal of the current study was to assess the effects of Psgl-1 deficiency on perivascular adipose tissue (PVAT) macrophage accumulation and endothelial dysfunction in the setting of obesity. Methods: Psgl-1 deficient ( Psgl-1 -/- ) and control ( Psgl-1 +/+ ) mice were fed either normal or DIO chow for 10 weeks (n=5-7 mice per group). Mesenteric arterioles were then mounted on a pressure myograph to evaluate vascular function. Norepinephrine (NE)-induced vasoconstriction, acetylcholine (Ach)-induced (with and without N-nitro-L-arginine methyl ester (L-NAME)) and sodium nitroprusside (SNP)-induced vasorelaxation were evaluated. Mesenteric PVAT macrophage content was assessed with Mac-3 immunohistochemistry. Results: The body weight of DIO mice was greater than standard chow-fed mice (46.5±1.4 vs 21.2±1.6g, p<0.01). NE-induced vasoconstrictions were similar between Psgl-1 +/+ and Psgl-1 -/- mice on DIO or standard chow, however, relaxation responses to Ach were significantly impaired in DIO Psgl-1 +/+ mice compared with standard chow-fed Psgl-1 +/+ mice (maximal relaxation, 36.0±7.8% vs 73.4±5.3%, p<0.01). In contrast Psgl-1 -/- DIO mice were protected from endothelial dysfunction and were similar to Psgl-1 -/- mice on standard chow (maximal relaxation, 75.1±4.8% and 83.8±3.7%). Ach-induced vasorelaxation was completely inhibited in all groups in the presence of L-NAME. Endothelium-independent relaxation responses to SNP were similar in all groups. Mac-3 staining showed reduced macrophage content in mesenteric PVAT of DIO Psgl-1 -/- mice compared to DIO Psgl-1 +/+ mice (18.2±1.5% vs 30.0±2.6% , p<0.01). Treatment of DIO Psgl-1 +/+ mice with a Psgl-1 antibody for 4 weeks resulted in Ach-induced relaxation of 71.6±10.9%, which was similar to Psgl-1 -/- DIO mice. Conclusions: Endothelial function and vascular NO bioavailability are impaired in a DIO mouse model. Psgl-1 deficiency is protective against PVAT inflammation and endothelial dysfunction induced by obesity. Psgl-1 inhibition may be useful for treatment of obesity-related vascular diseases.

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.

Lack of P-selectin glycoprotein ligand-1 protects mice from thrombosis after collagen/epinephrine challenge

IntroductionIn thrombotic processes, during the association of leukocytes with platelets and endothelial cells, P-selectin glycoprotein ligand-1 (PSGL-1) binds to P-selectin, expressedon activated platelets and endothelial cells. Our aim was to establish the role of PSGL-1 in thrombus formation by evaluating the response to thrombotic stimuli in wild type and PSGL-1 knockout mice. Materials and methodsMice were challenged by tail vein injection of (i) 15μg collagen plus 3μg epinephrine (coll/epi) (ii) 7.5μg collagen plus 1.5μg epinephrine or (iii) saline. Retro-orbital blood samples were collected in ACD anticoagulaed tubes and platelet and leukocyte counts were measured. In addition, kidneys, liver, spleen and lungs were investigated for fibrin deposition by immunohistochemistry and Western-blotting. Frozen sections were analysed for double labeling for platelet and leukocyte presence. ResultsAfter coll/epi challenge, the number of platelets and leukocytes decreased significantly in both genotypes. Lower agonist concentration resulted in an attenuated platelet decrease in PSGL-1 knockout mice compared to the controls, however changes in leukocyte and neutrophil counts were not significantly different in the two strains. In knockout mice considerably less fibrin deposition has been observedin the lungs by Western-blotting and immunohistochemistry. After coll/epi challenge the lungs of the PSGL-1 knockout animals contained both platelets and leukocytes but less thrombi has been detected than in wild-type mice. ConclusionsOur results indicate that the deficiency of PSGL-1 results in milder thrombocytopenia, less fibrin deposition and lower number of thrombosed blood vessels, suggesting that this molecule is essential for multicellular interactions during thrombus formation.

P-selectin glycoprotein ligand-1 deficiency is protective against obesity-related insulin resistance

P-selectin glycoprotein ligand-1 deficiency is protective against obesity-related insulin resistance

P-Selectin Glycoprotein Ligand-1 Negatively Regulates T-Cell Immune Responses

Cell surface sialomucins often act as antiadhesive molecules by virtue of their extended structure and negative charge. CD43 is one such sialomucin, expressed on most leukocytes. P-selectin glycoprotein ligand-1 (PSGL-1) is another sialomucin expressed by leukocytes. It serves as a major selectin ligand, but no antiadhesive role for it has been described. In this study, we showed that PSGL-1-deficient T cells, like CD43-deficient T cells, exhibited increased adhesion and proliferation compared with wild-type cells. The loss of both PSGL-1 and CD43 led to a further increase in T cell adhesion and proliferation. The reexpression of full-length PSGL-1 or CD43 in double-deficient CD4(+) T cells reversed their increased adhesion and proliferation phenotype. Using chimeric constructs of human CD8 and either PSGL-1 or CD43, we demonstrated that the intracellular domain of PSGL-1 or CD43 is required for suppressing proliferation but not adhesion. Furthermore, in a mouse model of inflammatory bowel disease induced by the adoptive transfer of naive T cells into RAG-deficient hosts, a PSGL-1 deficiency exacerbated the development of inflammation. These results reveal a novel regulatory role for PSGL-1 in T cell adhesion and proliferation and suggest that PSGL-1 negatively regulates T cell immune responses in vivo.

P-Selectin Glycoprotein Ligand-1 Contributes to Wound Healing Predominantly as a P-Selectin Ligand and Partly as an E-Selectin Ligand

Cell adhesion molecules are critical to wound healing through leukocyte recruitment. Although P-selectin glycoprotein ligand-1 (PSGL-1) regulates leukocyte rolling by binding P-selectin, but also binding E- and L-selectins with lower affinity, little is known about a role of PSGL-1 in wound healing. To clarify a role of PSGL-1 and its interaction with E- and P-selectins in wound healing, we investigated cutaneous wound healing in PSGL-1-deficient (PSGL-1(-/-)) mice in comparison with E-selectin(-/-), P-selectin(-/-), and P-selectin(-/-) mice treated with an anti-E-selectin antibody. PSGL-1 deficiency inhibited early wound healing, which was accompanied by decreased inflammatory cell infiltration and growth factor expression. By contrast, E-selectin deficiency did not affect wound healing. In general, the inhibitory effect of PSGL-1 deficiency on wound healing was similar to that of P-selectin deficiency either alone or with E-selectin blockade. However, early granulation tissue formation, late angiogenesis, and early infiltration of neutrophils and macrophages in PSGL-1(-/-) mice were inhibited beyond the inhibition in P-selectin(-/-) mice, but to a similar level of inhibition in P-selectin(-/-) mice with E-selectin blockade. These results suggest that PSGL-1 contributes to wound healing predominantly as a P-selectin ligand and partly as an E-selectin ligand by mediating infiltration of inflammatory cells.

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.

CD43 Collaborates with P-Selectin Glycoprotein Ligand-1 to Mediate E-Selectin-Dependent T Cell Migration into Inflamed Skin

Activated T cell migration into nonlymphoid tissues is initiated by the interactions of P- and E-selectin expressed on endothelial cells and their ligands on T cells. P-selectin glycoprotein ligand-1 (PSGL-1) has been the only E-selectin ligand demonstrated to function during the in vivo migration of activated T cells. We show in this study that CD43-deficient Th1 cells, like PSGL-1-deficient cells, exhibited reduced E-selectin-binding activity compared with wild-type cells. Th1 cells with a PSGL-1 and CD43 double deficiency showed even less E-selectin-binding activity. In migration assays in which adoptively transferred cells migrate to inflamed skin P- and E-selectin dependently, CD43 contributed significantly to PSGL-1-independent Th1 cell migration. In addition, in vivo activated T cells from the draining lymph nodes of sensitized mice deficient in PSGL-1 and/or CD43 showed significantly decreased E-selectin-binding activity and migration efficiency, with T cells from double-deficient mice showing the most profound decrease. Collectively, these results demonstrate that the CD43 expressed on activated T cells functions as an E-selectin ligand and thereby mediates T cell migration to inflamed sites, in collaboration with PSGL-1.