Bromelain decreases neutrophil interactions with P-selectin, but not E-selectin, in vitro by proteolytic cleavage of P-selectin glycoprotein ligand-1.
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ABSTRACT:
SUBMITTER: Banks JM
PROVIDER: S-EPMC3823987 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Bromelain, a mixture of proteases derived from pineapple stem, has been reported to have therapeutic benefits in a variety of inflammatory diseases, including murine inflammatory bowel disease. The purpose of this work was to understand potential mechanisms for this anti-inflammatory activity. Exposure to bromelain in vitro has been shown to remove a number of cell surface molecules that are vital to leukocyte trafficking, including CD128a/CXCR1 and CD128b/CXCR2 that serve as receptors for the neutrophil chemoattractant IL-8 and its murine homologues. We hypothesized that specific proteolytic removal of CD128 molecules by bromelain would inhibit neutrophil migration to IL-8 and thus decrease acute responses to inflammatory stimuli. Using an in vitro chemotaxis assay, we demonstrated a 40% reduction in migration of bromelain- vs. sham-treated human neutrophils in response to rhIL-8. Migration to the bacterial peptide analog fMLP was unaffected, indicating that bromelain does not induce a global defect in leukocyte migration. In vivo bromelain treatment generated a 50-85% reduction in neutrophil migration in 3 different murine models of leukocyte migration into the inflamed peritoneal cavity. Intravital microscopy demonstrated that although in vivo bromelain treatment transiently decreased leukocyte rolling, its primary long-term effect was abrogation of firm adhesion of leukocytes to blood vessels at the site of inflammation. These changes in adhesion were correlated with rapid re-expression of the bromelain-sensitive CD62L/L-selectin molecules that mediate rolling following in vivo bromelain treatment and minimal re-expression of CD128 over the time period studied. Taken together, these studies demonstrate that bromelain can effectively decrease neutrophil migration to sites of acute inflammation and support the specific removal of the CD128 chemokine receptor as a potential mechanism of action.
| S-EPMC2516972 | biostudies-literature
Project description: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.
| S-EPMC2597599 | biostudies-literature
Project description:Acute inflammation begins with leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) binding to P-selectin on inflamed endothelium and platelets. In pathologic conditions, this process may contribute to secondary organ damage, like sepsis-induced liver injury. Therefore, developing novel therapies to attenuate inflammation may be beneficial. We previously reported that recombinant human vimentin (rhVim) binds P-selectin to block leukocyte adhesion to endothelium and platelets. In this study, we used SPOT-peptide arrays to identify the rod domain as the active region within rhVim that interacts with P-selectin. Indeed, recombinant human rod domain of vimentin (rhRod) binds to P-selectin with high affinity, with in silico modeling suggesting that rhRod binds P-selectin at or near the PSGL-1 binding site. Using bio-layer interferometry, rhRod decreases PSGL-1 binding to immobilized P-selectin, corroborating the in silico data. Under parallel-plate flow, rhRod blocks leukocyte adhesion to fibrin(ogen)-captured platelets, P-selectin/Fc-coated channels, and IL-1?/IL-4-co-stimulated human umbilical vein endothelial cells. Finally, using intravital microscopy in endotoxemic C57Bl/6 mice, rhRod co-localizes with P-selectin in the hepatic sinusoids and decreases neutrophil adhesion to hepatic sinusoids. These data suggest a potential role for rhRod in attenuating inflammation through directly blocking P-selectin-PSGL-1 interactions.
| S-EPMC7553327 | biostudies-literature
Project description:PURPOSE:Obstructive sleep apnea (OSA) is characterized by chronic intermittent hypoxia which induces inflammation in blood vessels leading to the development of cardiovascular comorbidities. Several studies implicated the role of P-selectin in vascular inflammation of OSA. P-selectin glycoprotein ligand 1 (PSGL-1) is the main activator for P-selectin and is involved in immune cell trafficking. However, PSGL-1 has not been analyzed in OSA. The aim of the study was to investigate plasma PSGL-1 and P-selectin levels to have a deeper understanding on their interaction in obstructive sleep apnea. METHODS:Fifty-one untreated patients with OSA and 42 non-OSA controls were recruited. Plasma PSGL-1 levels were determined in evening and morning samples, P-selectin levels were analyzed in morning samples using commercially available ELISA kits. Polysomnography was performed in all participants. OSA was defined by an apnea-hypopnea index???5/h. RESULTS:PSGL-1 levels did not differ between controls and OSA patients either in the evening or in the morning. Although, there was no difference between controls (16.9/6.8-40.8 ng/ml) and patients with OSA (19.6/8.4-56.8, p?=?0.24), patients with severe OSA had increased plasma P-selectin levels (25.6/8.4-56.8 ng/ml) compared to mild OSA patients (14.1/8.5-35.3 ng/ml, p?=?0.006) and controls (p?=?0.03). CONCLUSIONS:P-selectin expression relates to disease severity suggesting a pathophysiological role in endothelial cell activation. PSGL-1 levels are unaltered in OSA, suggesting an alternative activation pathway for P-selectin in OSA.
| S-EPMC7012996 | biostudies-literature
Project description:IntroductionThe glycocalyx is a layer of glycoproteins, proteoglycans and glycosaminoglycans that coats the luminal surface of most blood vessels. It effectively regulates adhesive interactions between leukocytes in flowing blood and the endothelium, where during inflammation, binding to E- and P-selectins and intercellular adhesion molecule-1 (ICAM-1) promotes cell tethering and arrest under shear flow.MethodsIn this study, we examine the targeting of E-selectin by an engineered peptide moiety bound to a dermatan sulfate backbone. We further investigate this conjugate, denoted as EC-SEAL, by observing its binding to inflamed endothelium, and quantifying its ability to modulate neutrophil-endothelium interactions.ResultsBinding data reveal that EC-SEAL recognizes domains on E-selectin, and to a lesser degree on P- and L-selectin, and ICAM-1. Further, EC-SEAL increases neutrophil rolling velocity, and decreases neutrophil arrest and migration on inflamed human microvascular endothelial cells under physiologically relevant flow conditions.ConclusionsWe conclude that simple targeting strategies can mimic glycocalyx function under inflammatory conditions, effectively reducing neutrophil recruitment.
| S-EPMC6345733 | biostudies-literature
Project description:BACKGROUND: P-selectin glycoprotein ligand-1 (PSGL-1) plays a critical role in recruiting leukocytes in inflammatory lesions by mediating leukocyte rolling on selectins. Core-2 O-glycosylation of a N-terminal threonine and sulfation of at least one tyrosine residue of PSGL-1 are required for L- and P-selectin binding. Little information is available on the intra- and inter-species evolution of PSGL-1 primary structure. In addition, the evolutionary conservation of selectin binding site on PSGL-1 has not been previously examined in detail. Therefore, we performed multiple sequence alignment of PSGL-1 amino acid sequences of 14 mammals (human, chimpanzee, rhesus monkey, bovine, pig, rat, tree-shrew, bushbaby, mouse, bat, horse, cat, sheep and dog) and examined mammalian PSGL-1 interactions with human selectins. RESULTS: A signal peptide was predicted in each sequence and a propeptide cleavage site was found in 9/14 species. PSGL-1 N-terminus is poorly conserved. However, each species exhibits at least one tyrosine sulfation site and, except in horse and dog, a T [D/E]PP [D/E] motif associated to the core-2 O-glycosylation of a N-terminal threonine. A mucin-like domain of 250-280 amino acids long was disclosed in all studied species. It lies between the conserved N-terminal O-glycosylated threonine (Thr-57 in human) and the transmembrane domain, and contains a central region exhibiting a variable number of decameric repeats (DR). Interspecies and intraspecies polymorphisms were observed. Transmembrane and cytoplasmic domain sequences are well conserved. The moesin binding residues that serve as adaptor between PSGL-1 and Syk, and are involved in regulating PSGL-1-dependent rolling on P-selectin are perfectly conserved in all analyzed mammalian sequences. Despite a poor conservation of PSGL-1 N-terminal sequence, CHO cells co-expressing human glycosyltransferases and human, bovine, pig or rat PSGL-1 efficiently rolled on human L- or P-selectin. By contrast, pig or rat neutrophils were much less efficiently recruited than human or bovine neutrophils on human selectins. Horse PSGL-1, glycosylated by human or equine glycosyltransferases, did not interact with P-selectin. In all five species, tyrosine sulfation of PSGL-1 was required for selectin binding. CONCLUSION: These observations show that PSGL-1 amino acid sequence of the transmembrane and cytoplasmic domains are well conserved and that, despite a poor conservation of PSGL-1 N-terminus, L- and P-selectin binding sites are evolutionary conserved. Functional assays reveal a critical role for post-translational modifications in regulating mammalian PSGL-1 interactions with selectins.
| S-EPMC2174952 | biostudies-literature
Project description:P-selectin glycoprotein ligand-1 (PSGL-1) is a membrane-bound glycoprotein expressed in lymphoid and myeloid cells. It is a ligand of P-, E- and L-selectin and is involved in T cell trafficking and homing to lymphoid tissues, among other functions. PSGL-1 expression has been implicated in different lymphoid malignancies, so here we aimed to evaluate the involvement of PSGL-1 in T cell lymphomagenesis and dissemination. PSGL-1 was highly expressed at the surface of human and mouse T cell leukemia and lymphoma cell lines. To assess its impact on T cell malignancies, we stably expressed human PSGL-1 (hPSGL-1) in a mouse thymic lymphoma cell line, which expresses low levels of endogenous PSGL-1 at the cell surface. hPSGL-1-expressing lymphoma cells developed subcutaneous tumors in athymic nude mice recipients faster than control empty vector or parental cells. Moreover, the kidneys, lungs and liver of tumor-bearing mice were infiltrated by hPSGL-1-expressing malignant T cells. To evaluate the role of PSGL-1 in lymphoma cell dissemination, we injected intravenously control and hPSGL-1-expressing lymphoma cells in athymic mice. Strikingly, PSGL-1 expression facilitated disease infiltration of the kidneys, as determined by histological analysis and anti-CD3 immunohistochemistry. Together, these results indicate that PSGL-1 expression promotes T cell lymphoma development and dissemination to different organs.
| S-EPMC8188565 | biostudies-literature
Project description:The mature envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) virions is derived by proteolytic cleavage of a trimeric gp160 glycoprotein precursor. Remarkably, proteolytic processing of the HIV-1 Env precursor results in changes in Env antigenicity that resemble those associated with glutaraldehyde fixation. Apparently, proteolytic processing of the HIV-1 Env precursor decreases conformational flexibility of the Env trimeric complex, differentially affecting the integrity/accessibility of epitopes for neutralizing and nonneutralizing antibodies.
| S-EPMC3554131 | biostudies-literature
Project description:Bacterial and viral respiratory infections can initiate acute lung injury and acute respiratory distress syndrome. Neutrophils and their granule enzymes, including neutrophil elastase, are key mediators of the pathophysiology of acute respiratory failure. Although intracellular neutrophil elastase functions as a host defensive factor against pathogens, its leakage into airway spaces induces degradation of host connective tissue components. This leakage disrupts host innate immune responses via proteolytic cleavage of Toll-like receptors and cytokines. Here, we investigated whether neutrophils possess proteases that cleave adaptive immune molecules. We found that expression of the human leukocyte antigen (HLA) class II molecule HLA-DP β1 was decreased in THP-1-derived macrophages treated with supernatants from dead neutrophils. This decreased HLA-DP β1 expression was counteracted by treatment with neutrophil elastase inhibitor, suggesting proteolytic cleavage of HLA-DP β1 by neutrophil elastase. SDS-PAGE showed that neutrophil elastase cleaved recombinant HLA-DP α1, -DP β1, -DQ α1, -DQ β1, -DR α, and -DR β1. Neutrophil elastase also cleaved HLA-DP β1 on extracellular vesicles isolated from macrophages without triggering morphological changes. Thus, leakage of neutrophil elastase may disrupt innate immune responses, antigen presentation, and T cell activation. Additionally, inhibition of neutrophil elastase is a potential therapeutic option for treating bacterial and viral pneumonia.
| S-EPMC7843615 | biostudies-literature
Project description:OBJECTIVE: An inflammatory process is involved in the mechanism of obesity-related insulin resistance. Recent studies indicate that monocyte chemoattractant protein-1 (MCP-1) is a major chemokine that promotes monocyte infiltration into adipose tissues; however, the adhesion pathway in adipose tissues remains unclear. We aimed to clarify the adhesion molecules that mediate monocyte infiltration into adipose tissue. RESEARCH DESIGN AND METHODS: We used a DNA microarray to compare the gene expression profiles in epididymal white adipose tissues (eWAT) between db/db mice and C57/BL6 mice each fed a high-fat diet (HFD) or a low-fat diet (LFD). We investigated the change of insulin resistance and inflammation in eWAT in P-selectin glycoprotein ligand-1 (PSGL-1) homozygous knockout (PSGL-1?(/)?) mice compared with wild-type (WT) mice fed HFD. RESULTS: DNA microarray analysis revealed that PSGL-1, a major ligand for selectins, is upregulated in eWAT from both db/db mice and WT mice fed HFD. Quantitative real-time RT-PCR and immunohistochemistry showed that PSGL-1 is expressed on both endothelial cells and macrophages in eWAT of obese mice. PSGL-1?(/)? mice fed HFD showed a remarkable reduction of macrophage accumulation and expression of proinflammatory genes, including MCP-1 in eWAT. Moreover, adipocyte hypertrophy, insulin resistance, lipid metabolism, and hepatic fatty change were improved in PSGL-1?(/) ?mice compared with WT mice fed HFD. CONCLUSIONS: These results indicate that PSGL-1 is a crucial adhesion molecule for the recruitment of monocytes into adipose tissues in obese mice, making it a candidate for a novel therapeutic target for the prevention of obesity-related insulin resistance.
| S-EPMC3012171 | biostudies-literature