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1.
Immunology ; 157(2): 173-184, 2019 06.
Article in English | MEDLINE | ID: mdl-31013364

ABSTRACT

Leucocyte recruitment is critical during many acute and chronic inflammatory diseases. Chemokines are key mediators of leucocyte recruitment during the inflammatory response, by signalling through specific chemokine G-protein-coupled receptors (GPCRs). In addition, chemokines interact with cell-surface glycosaminoglycans (GAGs) to generate a chemotactic gradient. The chemokine interleukin-8/CXCL8, a prototypical neutrophil chemoattractant, is characterized by a long, highly positively charged GAG-binding C-terminal region, absent in most other chemokines. To examine whether the CXCL8 C-terminal peptide has a modulatory role in GAG binding during neutrophil recruitment, we synthesized the wild-type CXCL8 C-terminal [CXCL8 (54-72)] (Peptide 1), a peptide with a substitution of glutamic acid (E) 70 with lysine (K) (Peptide 2) to increase positive charge; and also, a scrambled sequence peptide (Peptide 3). Surface plasmon resonance showed that Peptide 1, corresponding to the core CXCL8 GAG-binding region, binds to GAG but Peptide 2 binding was detected at lower concentrations. In the absence of cellular GAG, the peptides did not affect CXCL8-induced calcium signalling or neutrophil chemotaxis along a diffusion gradient, suggesting no effect on GPCR binding. All peptides equally inhibited neutrophil adhesion to endothelial cells under physiological flow conditions. Peptide 2, with its greater positive charge and binding to polyanionic GAG, inhibited CXCL8-induced neutrophil transendothelial migration. Our studies suggest that the E70K CXCL8 peptide, may serve as a lead molecule for further development of therapeutic inhibitors of neutrophil-mediated inflammation based on modulation of chemokine-GAG binding.


Subject(s)
Cell Adhesion/immunology , Cell Movement/immunology , Endothelial Cells/immunology , Interleukin-8/immunology , Neutrophils/immunology , Endothelial Cells/pathology , Humans , Inflammation/immunology , Inflammation/pathology , Neutrophils/pathology , Peptides/immunology
2.
Int J Nanomedicine ; 7: 2943-56, 2012.
Article in English | MEDLINE | ID: mdl-22745555

ABSTRACT

Vascular endothelium is a potential target for therapeutic intervention in diverse pathological processes, including inflammation, atherosclerosis, and thrombosis. By virtue of their intravascular topography, endothelial cells are exposed to dynamically changing mechanical forces that are generated by blood flow. In the present study, we investigated the interactions of negatively charged 2.7 nm and 4.7 nm CdTe quantum dots and 50 nm silica particles with cultured endothelial cells under regulated shear stress (SS) conditions. Cultured cells within the engineered microfluidic channels were exposed to nanoparticles under static condition or under low, medium, and high SS rates (0.05, 0.1, and 0.5 Pa, respectively). Vascular inflammation and associated endothelial damage were simulated by treatment with tumor necrosis factor-α (TNF-α) or by compromising the cell membrane with the use of low Triton X-100 concentration. Our results demonstrate that SS is critical for nanoparticle uptake by endothelial cells. Maximal uptake was registered at the SS rate of 0.05 Pa. By contrast, endothelial exposure to mild detergents or TNF-α treatment had no significant effect on nanoparticle uptake. Atomic force microscopy demonstrated the increased formation of actin-based cytoskeletal structures, including stress fibers and membrane ruffles, which have been associated with nanoparticle endocytosis. In conclusion, the combinatorial effects of SS rates, vascular endothelial conditions, and nanoparticle physical and chemical properties must be taken into account for the successful design of nanoparticle-drug conjugates intended for parenteral delivery.


Subject(s)
Human Umbilical Vein Endothelial Cells/metabolism , Nanoparticles/chemistry , Quantum Dots , Actin Cytoskeleton/metabolism , Cadmium Compounds/chemistry , Cadmium Compounds/pharmacokinetics , Cell Growth Processes/physiology , Cell Membrane/metabolism , Humans , Inflammation/metabolism , Inflammation/pathology , Microfluidics , Microscopy, Atomic Force , Models, Biological , Octoxynol , Silicon Dioxide/chemistry , Silicon Dioxide/pharmacokinetics , Stress, Mechanical , Tellurium/chemistry , Tellurium/pharmacokinetics , Tumor Necrosis Factor-alpha
3.
J Immunol ; 188(12): 6357-70, 2012 Jun 15.
Article in English | MEDLINE | ID: mdl-22581862

ABSTRACT

Chemokines such as SDF-1α play a crucial role in orchestrating T lymphocyte polarity and migration via polymerization and reorganization of the F-actin cytoskeleton, but the role of actin-associated proteins in this process is not well characterized. In this study, we have investigated a role for L-plastin, a leukocyte-specific F-actin-bundling protein, in SDF-1α-stimulated human T lymphocyte polarization and migration. We found that L-plastin colocalized with F-actin at the leading edge of SDF-1α-stimulated T lymphocytes and was also phosphorylated at Ser(5), a site that when phosphorylated regulates the ability of L-plastin to bundle F-actin. L-plastin phosphorylation was sensitive to pharmacological inhibitors of protein kinase C (PKC), and several PKC isoforms colocalized with L-plastin at the leading edge of SDF-1α-stimulated lymphocytes. However, PKC ζ, an established regulator of cell polarity, was the only isoform that regulated L-plastin phosphorylation. Knockdown of L-plastin expression with small interfering RNAs demonstrated that this protein regulated the localization of F-actin at the leading edge of chemokine-stimulated cells and was also required for polarization, lamellipodia formation, and chemotaxis. Knockdown of L-plastin expression also impaired the Rac1 activation cycle and Akt phosphorylation in response to SDF-1α stimulation. Furthermore, L-plastin also regulated SDF-1α-mediated lymphocyte migration on the integrin ligand ICAM-1 by influencing velocity and persistence, but in a manner that was independent of LFA-1 integrin activation or adhesion. This study, therefore, demonstrates an important role for L-plastin and the signaling pathways that regulate its phosphorylation in response to chemokines and adds L-plastin to a growing list of proteins implicated in T lymphocyte polarity and migration.


Subject(s)
Cell Polarity/immunology , Chemotaxis, Leukocyte/immunology , Membrane Glycoproteins/metabolism , Microfilament Proteins/metabolism , Signal Transduction/immunology , T-Lymphocytes/metabolism , Actins/immunology , Actins/metabolism , Blotting, Western , Chemokines/immunology , Chemokines/metabolism , Cytoskeleton/immunology , Cytoskeleton/metabolism , Flow Cytometry , Fluorescent Antibody Technique , Gene Knockdown Techniques , Humans , Lymphocyte Activation/immunology , Membrane Glycoproteins/immunology , Microfilament Proteins/immunology , Phosphorylation , RNA, Small Interfering , T-Lymphocytes/immunology
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