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The physiologic role of L-selectin shedding is unknown. Here, we investigate the effect of L-selectin shedding on firm adhesion and transmigration. In a tumor necrosis factor alpha-induced model of inflammation, inhibition of L-selectin shedding significantly increased firm adhesion and transmigration by a lymphocyte function-associated antigen (LFA)-1 and intercellular adhesion molecule (ICAM)-1-dependent mechanism. We examined the quality of leukocyte rolling and L-selectin-mediated signaling. Blockade of L-selectin shedding significantly reduced the "jerkiness" of leukocyte rolling, defined as the variability of velocity over time. A low level of jerkiness was also observed in the rolling of microbeads conjugated with L-selectin, a model system lacking the mechanism for L-selectin shedding. Inhibition of L-selectin shedding potentiated activation of LFA-1 and Mac-1 induced by L-selectin cross-linking as shown by activation epitope expression and binding of ICAM-1-conjugated beads. We conclude that inhibition of L-selectin shedding increases leukocyte adhesion and transmigration by (a) increasing leukocyte exposure to the inflamed endothelium by decreasing jerkiness and (b) promoting leukocyte activation by outside-in signaling. These observations help to resolve the apparent discrepancy between the minor contribution of L-selectin to rolling and the significant leukocyte recruitment defect in L-selectin knockout mice.
L-selectin mediates the initial tethering and rolling of lymphocytes in high endothelial venules. To study the transcriptional regulation of mouse L-selectin, we cloned 4.5 kb 5'-flanking sequences of the mouse sell. Luciferase analysis of serial 5'-deletion mutants showed that the first 285 bp was sufficient to drive high promoter activity in EL4 cells, but not in Sell-negative HeLa cells, suggesting that this fragment harbors the minimal mouse sell promoter and contains cis-elements for lymphocyte-specific expression. Site-directed mutagenesis and chromatin immunoprecipitation showed that Mzf1, Klf2, Sp1, Ets1, and Irf1 bind to and activate the mouse sell promoter. Over expression of these transcription factors in EL4 cells increased expression of sell mRNA. Silencing the expression of Sp1 by siRNA significantly decreased sell promoter activity in EL4 cells. We conclude that sell transcription is regulated by Mzf1, Klf2, Sp1, Ets1, and Irf1.
The calmodulin (CaM) hypothesis of ectodomain shedding stipulates that CaM, an intracellular Ca²⁺-dependent regulatory protein, associates with the cytoplasmic domain of L-selectin to regulate ectodomain shedding of L-selectin on the other side of the plasma membrane. To understand the underlying molecular mechanism, we have characterized the interactions of CaM with two peptides derived from human L-selectin. The peptide ARR18 corresponds to the entire cytoplasmic domain of L-selectin (residues Ala317-Tyr334 in the mature protein), and CLS corresponds to residues Lys280-Tyr334, which contains the entire transmembrane and cytoplasmic domains of l-selectin. Monitoring the interaction by fluorescence spectroscopy and other biophysical techniques, we found that CaM can bind to ARR18 in aqueous solutions or the L-selectin cytoplasmic domain of CLS reconstituted in the phosphatidylcholine bilayer, both with an affinity of approximately 2 μM. The association is calcium independent and dynamic and involves both lobes of CaM. In a phospholipid bilayer, the positively charged L-selectin cytoplasmic domain of CLS is associated with anionic phosphatidylserine (PS) lipids at the membrane interface through electrostatic interactions. Under conditions where the PS content mimics that in the inner leaflet of the cell plasma membrane, the interaction between CaM and CLS becomes undetectable. These results suggest that the association of CaM with L-selectin in the cell can be influenced by the membrane bilayer and that anionic lipids may modulate ectodomain shedding of transmembrane receptors.
The pleiotropic effects of statins might involve preventing inflammatory cell adhesion to the endothelium, which is a critical step in the pathogenesis of atherosclerosis. We conducted a systematic review and meta-analysis of the effects of statins on the circulating cell adhesion molecules E-Selectin, L-Selectin, and P-Selectin. A literature search was conducted in PubMed, Web of Science, and Scopus, from inception to July 2021. Risk of bias and certainty of evidence were assessed using the Joanna Briggs Institute Critical Appraisal Checklist and GRADE, respectively. In 61 studies, statins significantly reduced P-selectin (standard mean difference, SMD = -0.39, 95% CI -0.55 to -0.22, p < 0.001; moderate certainty of evidence), L-selectin (SMD = -0.49, 95% CI -0.89 to -0.10, p = 0.014; very low certainty of evidence), and E-Selectin (SMD = -0.73, 95% CI -1.02 to -0.43, p < 0.001; moderate certainty of evidence), independently of baseline lipid profile and other study and patient characteristics. The corresponding pooled SMD values in sensitivity analysis were not substantially altered when individual studies were sequentially removed. Simvastatin had a significant lowering effect on both P-selectin and E-selectin. Therefore, statins significantly reduce circulating selectins. Further studies are required to investigate whether selectin lowering mediates cardiovascular risk reduction with these agents. (PROSPERO registration number: CRD42021282778).
The migration of circulating neutrophils towards damaged or infected tissue is absolutely critical to the inflammatory response. L-selectin is a cell adhesion molecule abundantly expressed on circulating neutrophils. For over two decades, neutrophil L-selectin has been assigned the exclusive role of supporting tethering and rolling - the initial stages of the multi-step adhesion cascade. Here, we provide direct evidence for L-selectin contributing to neutrophil transendothelial migration (TEM). We show that L-selectin co-clusters with PECAM-1 - a well-characterised cell adhesion molecule involved in regulating neutrophil TEM. This co-clustering behaviour occurs specifically during TEM, which serves to augment ectodomain shedding of L-selectin and expedite the time taken for TEM (TTT) to complete. Blocking PECAM-1 signalling (through mutation of its cytoplasmic tail), PECAM-1-dependent adhesion or L-selectin shedding, leads to a significant delay in the TTT. Finally, we show that co-clustering of L-selectin with PECAM-1 occurs specifically across TNF- but not IL-1β-activated endothelial monolayers - implying unique adhesion interactomes forming in a cytokine-specific manner. To our knowledge, this is the first report to implicate a non-canonical role for L-selectin in regulating neutrophil TEM.
Multivalent interactions occur frequently in nature, where they mediate high-affinity interactions between cells, proteins, or molecules. Here, we report on a method to generate multivalent aptamers (Multi-Aptamers) that target L-selectin function using rolling circle amplification (RCA). We find that the L-selectin Multi-Aptamers have increased affinity compared to the monovalent aptamer, are specific to L-selectin, and are capable of inhibiting interactions with endogenous ligands. In addition, the Multi-Aptamers efficiently inhibit L-selectin mediated dynamic adhesion in vitro and homing to secondary lymphoid tissues in vivo. Importantly, our method of generating multivalent materials using RCA avoids many of the challenges associated with current multivalent materials in that the Multi-Aptamers are high affinity, easily produced and modified, and biocompatible. We anticipate that the Multi-Aptamers can serve as a platform technology to modulate diverse cellular processes.
Circulating neutrophils must avoid premature activation to prevent tissue injury. The leukocyte adhesion receptor L-selectin forms bonds with P-selectin glycoprotein ligand-1 (PSGL-1) on other leukocytes and with peripheral node addressin (PNAd) on high endothelial venules. Mechanical forces can strengthen (catch) or weaken (slip) bonds between biological molecules. How these mechanochemical processes influence function in vivo is unexplored. Here we show that mice expressing an L-selectin mutant (N138G) have altered catch bonds and prolonged bond lifetimes at low forces. Basal lymphocyte homing and neutrophil recruitment to inflamed sites are normal. However, circulating neutrophils form unstable aggregates and are unexpectedly primed to respond robustly to inflammatory mediators. Priming requires signals transduced through L-selectin N138G after it engages PSGL-1 or PNAd. Priming enhances bacterial clearance but increases inflammatory injury and enlarges venous thrombi. Thus, L-selectin mechanochemistry limits premature activation of neutrophils. Our results highlight the importance of probing how mechanochemistry functions in vivo.
(1) L-selectin, constitutively expressed by leukocytes, is involved in the initial binding of leukocytes to activated endothelium. Anti-inflammatory drugs like glucocorticoids can induce shedding of L-selectin, but the mechanism is still unknown. Annexin 1, a protein whose synthesis and externalization/secretion are induced during the inflammatory response, has been proposed as a mediator of the anti-inflammatory actions of glucocorticoids. (2) The monocytic cell line U-937 strongly expresses Annexin 1 after 24 h of phorbol 12-myristate 13-acetate (PMA, 1 nm) treatment and externalizes/releases the protein after additional 16 h of dexamethasone (1 microm) treatment. (3) This study investigated the possible regulation of cell surface L-selectin shedding by endogenous Annexin 1, and its role in glucocorticoid-induced L-selectin shedding in the U-937 cell line. (4) PMA- and dexamethasone treatment-induced L-selectin shedding was potentially mediated by Annexin 1, since neutralizing antibodies against Annexin 1 reduced dexamethasone- and Annexin 1-induced shedding. (5) Immunoprecipitation and binding assays provided support for the suggestion that this effect could be mediated by an interaction between externalized Annexin 1 and L-selectin. Such interaction involved the N-terminal domain of Annexin 1 and was calcium-dependent. Confocal microscopy studies demonstrated increased colocalization of Annexin 1 and L-selectin on the cell surface. (6) Overall, our study provides new insights into the potential role of endogenous ANXA1 as a mediator of dexamethasone-induced L-selectin shedding, which may contribute to the anti-inflammatory activity of glucocorticoids.
L-selectin binding activity for its ligand expressed by vascular endothelium is rapidly and transiently increased after leukocyte activation. To identify mechanisms for upregulation and assess how this influences leukocyte/endothelial cell interactions, cell-surface dimers of L-selectin were induced using the coumermycin-GyrB dimerization strategy for cross-linking L-selectin cytoplasmic domains in L-selectin cDNA-transfected lymphoblastoid cells. Coumermycin- induced L-selectin dimerization resulted in an approximately fourfold increase in binding of phosphomanan monoester core complex (PPME), a natural mimic of an L-selectin ligand, comparable to that observed after leukocyte activation. Moreover, L-selectin dimerization significantly increased (by approximately 700%) the number of lymphocytes rolling on vascular endothelium under a broad range of physiological shear stresses, and significantly slowed their rolling velocities. Therefore, L-selectin dimerization may explain the rapid increase in ligand binding activity that occurs after leukocyte activation and may directly influence leukocyte migration to peripheral lymphoid tissues or to sites of inflammation. Inducible oligomerization may also be a common mechanism for rapidly upregulating the adhesive or ligand-binding function of other cell-surface receptors.
The velocity of rolling leukocytes is thought to be determined by the expression of adhesion molecules and the prevailing wall shear stress. Here, we investigate whether rapid cleavage of L-selectin may be an additional physiologic regulatory parameter of leukocyte rolling. A unique protease in the membrane of leukocytes cleaves L-selectin after activation, resulting in L-selectin shedding. The hydroxamic acid-based metalloprotease inhibitor KD-IX-73-4 completely prevented L-selectin shedding in vitro and significantly decreased the rolling velocity of leukocytes in untreated wild-type C57BL/6 mice from 55 to 35 micrometer/seconds in vivo. When E-selectin was expressed on the endothelium (tumor necrosis factor [TNF]-alpha treatment 2.5-3 h before the experiment), rolling velocity was 4 micrometer/seconds and did not change after the application of KD-IX-73-4. However, KD-IX-73-4 decreased mean rolling velocity by 29% from 23 to 16 micrometer/seconds in E-selectin-deficient mice treated with TNF-alpha. The reduction of velocity caused by KD-IX-73-4 was immediate (<5 s) after injection of KD-IX-73-4 as shown by a novel method using a local catheter. These results establish a role for L-selectin shedding in regulating leukocyte rolling velocity in vivo.
P-selectin expressed on activated endothelia and platelets supports recruitment of leukocytes expressing P-selectin ligand to sites of inflammation. While monitoring P-selectin ligand expression on activated CD8+ T cells in murine adoptive transfer models, we observed two distinct ligands on responding donor cells, the canonical cell-intrinsic P-selectin ligand PSGL-1 and a second undocumented P-selectin ligand we provisionally named PSL2. PSL2 is unusual among selectin ligands in that it is cell-extrinsic, loaded onto L-selectin expressed by activated T cells but not L-selectin on resting naïve CD8+ T cells. PSL2 display is highest on activated T cells responding in peripheral lymph nodes and low on T cells responding in spleen suggesting that the original source of PSL2 is high endothelial venules, cells known to produce L-selectin ligands. PSL2 is a ligand for both P-selectin and L-selectin and can physically bridge the two selectins. The L-selectin/PSL2 complex can mediate P-selectin-dependent adherence of activated T cells to immobilized P-selectin or to activated platelets, either independently or cooperatively with PSGL-1. PSL2's capacity to bridge between L-selectin on activated T cells and P-selectin reveals an undocumented and unanticipated activity of cell-extrinsic selectin ligands in mediating selectin-selectin connectivity. The timing and circumstances of PSL2 detection on T cells, together with its capacity to support adherence to P-selectin-bearing substrates, are consistent with P-selectin engagement of both PSGL1 and the L-selectin/PSL2 complex during T cell recruitment. Engagement of PSGL-1 and L-selectin/PSL2 would likely deliver distinct signals known to be relevant in this process.
Atherosclerosis is an inflammatory disease characterized by accumulation of leukocytes in the arterial intima. Members of the selectin family of adhesion molecules are important mediators of leukocyte extravasation. However, it is unclear whether L-selectin (L-sel) is involved in the pathogenesis of atherosclerosis. In the present study, mice deficient in L-selectin (L-sel(-/-)) animals were crossed with mice lacking Apolipoprotein E (ApoE(-/-)). The development of atherosclerosis was analyzed in double-knockout ApoE/L-sel (ApoE(-/-)L-sel(-/-)) mice and the corresponding ApoE(-/-) controls fed either a normal or a high cholesterol diet (HCD). After 6 weeks of HCD, aortic lesions were increased two-fold in ApoE(-/-)L-sel(-/-) mice as compared to ApoE(-/-) controls (2.46%±0.54% vs 1.28%±0.24% of total aortic area; p<0.05). Formation of atherosclerotic lesions was also enhanced in 6-month-old ApoE(-/-)L-sel(-/-) animals fed a normal diet (10.45%±2.58% vs 1.87%±0.37%; p<0.05). In contrast, after 12 weeks of HCD, there was no difference in atheroma formation between ApoE(-/-)L-sel(-/-) and ApoE(-/-) mice. Serum cholesterol levels remained unchanged by L-sel deletion. Atherosclerotic plaques did not exhibit any differences in cellular composition assessed by immunohistochemistry for CD68, CD3, CD4, and CD8 in ApoE(-/-)L-sel(-/-) as compared to ApoE(-/-) mice. Leukocyte rolling on lesions in the aorta was similar in ApoE(-/-)L-sel(-/-) and ApoE(-/-) animals. ApoE(-/-)L-sel(-/-) mice exhibited reduced size and cellularity of peripheral lymph nodes, increased size of spleen, and increased number of peripheral lymphocytes as compared to ApoE(-/-) controls. These data indicate that L-sel does not promote atherosclerotic lesion formation and suggest that it rather protects from early atherosclerosis.
The homing molecule, L-selectin (CD62L), is commonly used as a T cell activation marker, since expression is downregulated following engagement of the T cell receptor. Studies in mice have shown that CD62L+ central memory T cells are better at controlling tumor growth than CD62L- effector memory T cells, while L-selectin knockout T cells are poor at controlling tumor growth. Here, we test the hypothesis that T cells expressing genetically modified forms of L-selectin that are maintained following T cell activation (L-selectin enhanced T cells) are better at controlling tumor growth than wild type T cells. Using mouse models of adoptive cell therapy, we show that L-selectin enhancement improves the efficacy of CD8+ T cells in controlling solid and disseminated tumor growth. L-selectin knockout T cells had no effect. Checkpoint blockade inhibitors synergized with wild type and L-selectin enhanced T cells but had no effect in the absence of T cell transfers. Reduced tumor growth by L-selectin enhanced T cells correlated with increased frequency of CD8+ tumor infiltrating T cells 21 days after commencing therapy. Longitudinal tracking of Zirconium-89 (89Zr) labeled T cells using PET-CT showed that transferred T cells localize to tumors within 1 h and accumulate over the following 7 days. L-selectin did not promote T cell homing to tumors within 18 h of transfer, however the early activation marker CD69 was upregulated on L-selectin positive but not L-selectin knockout T cells. L-selectin positive and L-selectin knockout T cells homed equally well to tumor-draining lymph nodes and spleens. CD69 expression was upregulated on both L-selectin positive and L-selectin knockout T cells but was significantly higher on L-selectin expressing T cells, particularly in the spleen. Clonal expansion of isolated L-selectin enhanced T cells was slower, and L-selectin was linked to expression of proliferation marker Ki67. Together these findings demonstrate that maintaining L-selectin expression on tumor-specific T cells offers an advantage in mouse models of cancer immunotherapy. The beneficial role of L-selectin is unrelated to its' well-known role in T cell homing and, instead, linked to activation of therapeutic T cells inside tumors. These findings suggest that L-selectin may benefit clinical applications in T cell selection for cancer therapy and for modifying CAR-T cells to broaden their clinical scope.
A growing body of evidence suggests that L-selectin ligands presented on circulating tumor cells facilitate metastasis by binding L-selectin presented on leukocytes. Commonly used methods for detecting L-selectin ligands on tissues, e.g., immunostaining, are performed under static, no-flow conditions. However, such analysis does not assay for functional L-selectin ligands, specifically those ligands that promote adhesion under shear flow conditions. Recently our lab developed a method, termed dynamic biochemical tissue analysis (DBTA), to detect functional selectin ligands in situ by probing tissues with L-selectin-coated microspheres under hemodynamic flow conditions. In this investigation, DBTA was used to probe human colon tissues for L-selectin ligand activity. The detection of L-selectin ligands using DBTA was highly specific. Furthermore, DBTA reproducibly detected functional L-selectin ligands on diseased, e.g., cancerous or inflamed, tissues but not on noncancerous tissues. In addition, DBTA revealed a heterogeneous distribution of functional L-selectin ligands on colon cancer tissues. Most notably, detection of L-selectin ligands by immunostaining using HECA-452 antibody only partially correlated with functional L-selectin ligands detected by DBTA. In summation, the results of this study demonstrate that DBTA detects functional selectin ligands to provide a unique characterization of pathological tissue.
Neutrophils are recruited from the blood to sites of inflammation, where they contribute to immune defense but may also cause tissue damage. During inflammation, neutrophils roll along the microvascular endothelium before arresting and transmigrating. Arrest requires conformational activation of the integrin lymphocyte function-associated antigen 1 (LFA-1), which can be induced by selectin engagement. Here, we demonstrate that a subset of P-selectin glycoprotein ligand-1 (PSGL-1) molecules is constitutively associated with L-selectin. Although this association does not require the known lectin-like interaction between L-selectin and PSGL-1, the signaling output is dependent on this interaction and the cytoplasmic tail of L-selectin. The PSGL-1-L-selectin complex signals through Src family kinases, ITAM domain-containing adaptor proteins, and other kinases to ultimately result in LFA-1 activation. The PSGL-1-L-selectin complex-induced signaling effects on neutrophil slow rolling and recruitment in vivo demonstrate the functional importance of this pathway. We conclude that this is a signaling complex specialized for sensing adhesion under flow.
Immune dysfunction has been implicated in the pathophysiology of schizophrenia. Leukocyte migration to the site of inflammation is a fundamental step of immune response which involves P-, E-, and L-selectins. Elevated selectin levels have been reported in un-medicated first-episode patients with schizophrenia but not in medicated patients with multi-episode schizophrenia. We measured fasting plasma soluble P-, E-, and L-selectin in 39 medicated patients with multi-episode schizophrenia and 19 healthy controls. In patients, psychotic symptom severity and cognitive function were assessed with the Positive and Negative Syndrome Scale (PANSS) and the NIH Toolbox Cognitive Test Battery respectively. C-reactive protein (CRP) and Body Mass Index (BMI) were measured in patients and controls. Comparison of selectin levels between patients and controls was done with t-tests and linear regression. Pearson correlation coefficients between plasma selectins and PANSS and cognitive measures were calculated. Geometric mean plasma soluble L-selectin level was lower in patients compared to controls from unadjusted (606.7 ± 1.2 ng/ml vs. 937.7 ± 1.15 ng/ml, p < 0.001) and adjusted analyses (β = 0.59; CI 0.41 to 0.88, p = 0.011). There was a trend towards higher plasma soluble P-selectin in patients compared to controls (90.4 ± 1.2ng/ml vs. 71.8 ± 1.2ng/ml, p = 0.059) in the unadjusted analysis. There was no association between the selectins and psychotic symptoms or cognitive function in the patients. In addition, the selectins were not significantly associated with CRP or BMI. The limitations of this study include small sample size and unavailability of information on medications and blood cell counts. The potential utility of soluble L-selectin as a biomarker of antipsychotic exposure in patients with schizophrenia and the concomitant change in immune response with the use of antipsychotics should be further evaluated.
The leukocyte adhesion molecule L-selectin, which mediates the initial steps of leukocyte attachment to vascular endothelium, is intensely glycosylated. Different glycoforms of L-selectin are expressed on different leukocyte subsets and differences in L-selectin glycosylation appear to be correlated with the leukocyte's ability to attach to different endothelial targets. In the present study we addressed the question whether glycosylation of L-selectin influences L-selectin-ligand interactions. To obtain different glycoforms of L-selectin, recombinant proteins were expressed both in the baby hamster kidney (BHK) cell line and in the human myelogenous cell line K562, resulting in sL-sel[BHK] or sL-sel[K562], respectively. The glycosylation characteristics of the purified proteins were determined. The most striking differences in glycosylation were seen in the terminal sialylation. Each of the two proteins carried sialic acids in the alpha 2-3 position, while alpha 2-6-bound sialic acids were found exclusively on sL-sel[K562]. To investigate their adhesive properties, both recombinant sL-selectins were used in cell adhesion assays and interactions with the ligands present on various hematopoietic cell lines or activated human cardiac microvascular endothelial cells were examined. The binding capacity of sL-sel[K562] was about 1.6 fold higher compared to sL-sel[BHK] under static as well as under flow conditions. These findings indicate that the terminal sialylation pattern of L-selectin modulates its binding characteristics.
L-selectin is a cell adhesion molecule, which mediates leukocyte rolling on bronchopulmonary endothelium. Previous studies in a murine model of allergic airways disease have shown that L-selectin plays a role in the regulation of airway hyperresponsiveness in asthma via mechanisms independent of inflammation. Airway remodeling has been shown to modulate airway hyperresponsiveness independently of inflammation.
Peripheral lymph nodes (PLN) are critical for immunologic memory formation in response to antigens that penetrate the skin. Blood-borne lymphocytes first encounter such antigens after they home to PLN through a multi-step adhesion process that is normally initiated by L-selectin (CD62L) in high endothelial venules (HEV). Since naive T cells can not enter PLN normally in L-selectin-deficient mice, a delayed type hypersensitivity response to cutaneously applied antigen cannot be mounted. In this study, we report that the administration of activated platelets into the systemic circulation of L-selectin knockout mice restores lymphocyte trafficking to PLN, and reconstitutes T cell-mediated immunity in response to a cutaneous antigen. These effects required platelet-expressed P-selectin that allows activated platelets to transiently form a bridge between lymphocytes and HEV, thereby enabling lymphocytes to undergo subsequent beta2 integrin-dependent firm adhesion. These profound effects of platelet-mediated cell-cell interactions on lymphocyte trafficking and formation of immunologic memory may impact on a variety of autoimmune and inflammatory conditions.
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