interleukin-8 and 4-4-difluoro-4-bora-3a-4a-diaza-s-indacene

Hutchinson-Gilford progeria syndrome (HGPS) is a segmental premature aging disease caused by a mutation in

Topics: 3T3-L1 Cells; Adipocytes; Adipogenesis; Adolescent; Animals; Azetidines; Boron Compounds; Cell Differentiation; Cellular Senescence; Child; Child, Preschool; Cyclin-Dependent Kinase Inhibitor p16; Fatty Acid-Binding Proteins; Female; Fibroblasts; Humans; Interleukin-8; Janus Kinases; Lamin Type A; Male; Mice; PPAR gamma; Progeria; Protein Kinase Inhibitors; Purines; Pyrazoles; Skin; Stem Cells; Sulfonamides

Heparin is one of the most effective drugs for preventing and treating thromboembolic complications in surgical patients. Recent evidence suggests that heparin enhances the proinflammatory responses of human peripheral blood monocytes to Gram-negative endotoxin (LPS). We have identified LPS-binding protein (LBP) as a novel heparin-binding plasma protein. The affinity of LPB to heparin was KD = 55 +/- 8 nM, as measured by surface plasmon resonance. Using a fluorescence-based assay, we showed that clinically used heparin preparations significantly enhance the ability of LBP to catalytically disaggregate and transfer LPS to CD14, the LPS receptor. The presence of clinically relevant heparin concentrations in human whole blood increased LPS-induced production of the proinflammatory cytokine IL-8. Fondaparinux, which is identical with the antithrombin III-binding pentasaccharide in heparin, did not bind to LBP or alter LBP function. Thus, this novel anticoagulant drug is a potential candidate for safe administration to patients who have endotoxemia and require anticoagulation.

Topics: Acute-Phase Proteins; Adjuvants, Immunologic; Antithrombin III; Biological Transport; Boron Compounds; Carrier Proteins; Enoxaparin; Fluorescent Dyes; Heparin; Humans; Interleukin-8; Lipopolysaccharide Receptors; Lipopolysaccharides; Membrane Glycoproteins; Monocytes; Signal Transduction; Solubility; Sulfates; Teichoic Acids; Thrombin

Addition of lipopolysaccharide (LPS) to cells in the form of LPS-soluble (s)CD14 complexes induces strong cellular responses. During this process, LPS is delivered from sCD14 to the plasma membrane, and the cell-associated LPS is then rapidly transported to an intracellular site. This transport appears to be important for certain cellular responses to LPS, as drugs that block transport also inhibit signaling and cells from LPS-hyporesponsive C3H/HeJ mice fail to exhibit this transport. To identify the intracellular destination of fluorescently labeled LPS after its delivery from sCD14 into cells, we have made simultaneous observations of different organelles using fluorescent vital dyes or probes. Endosomes, lysosomes, the endoplasmic reticulum, and the Golgi apparatus were labeled using Texas red (TR)-dextran, LysoTrackertrade mark Red DND-99, DiOC6(3), and boron dipyrromethane (BODIPY)-ceramide, respectively. After 30 min, LPS did not colocalize with endosomes, lysosomes, or endoplasmic reticulum in polymorphonuclear leukocytes, although some LPS-positive vesicles overlapped with the endosomal marker, fluorescent dextran. On the other hand, LPS did appear to colocalize with two markers of the Golgi apparatus, BODIPY-ceramide and TRITC (tetramethylrhodamine isothiocyanate)-labeled cholera toxin B subunit. We further confirmed the localization of LPS in the Golgi apparatus using an epithelial cell line, HeLa, which responds to LPS-sCD14 complexes in a CD14-dependent fashion: BODIPY-LPS was internalized and colocalized with fluorescently labeled Golgi apparatus probes in live HeLa cells. Morphological disruption of the Golgi apparatus in brefeldin A-treated HeLa cells caused intracellular redistribution of fluorescent LPS. These results are consistent with the Golgi apparatus being the primary delivery site of monomeric LPS.

Topics: Biological Transport; Biomarkers; Boron Compounds; Cell Compartmentation; Cytoplasmic Granules; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Fluorescent Dyes; Golgi Apparatus; HeLa Cells; Humans; Interleukin-8; Lipopolysaccharide Receptors; Lipopolysaccharides; Lysosomes; Microscopy, Confocal; Molecular Probes; Neutrophils; Signal Transduction

Two G protein-coupled receptor subtypes (CXCR1 and CXCR2) mediate Interleukin-8 (IL8) action in cells. A nonradioactive lanthanide-chelate derivatized IL8 ligand was developed to measure the binding activity of the chemokine receptors, CXCR1 and CXCR2. Site-specific mutagenesis of the carboxyl-terminal serine of IL8 to cysteine resulted in a mutant IL8 (IL8-S72C) having a single free sulfhydryl. Using an iodoacetamide derivative of the Eu3+-chelate of N-(p-benzoic acid)diethylenetriamine-N,N',N"-tetraacetic acid (DTTA), incorporation of one Eu3+ per IL8 molecule ([Eu3+]IL8-S72C) was achieved. The dissociation constant for this conjugate was similar to that measured for [125I]IL8 ( approximately 2 nM) when measured by time-resolved fluorometry using CHO cell lines stably expressing CXCR1 or CXCR2 receptors. The sensitivity, stability, and high specific activity of europium-labeled IL8 demonstrate the usefulness of lanthanide-labeled proteins in the measurement of receptor-ligand interactions and may be extended to other peptide ligands.

Topics: Animals; Antigens, CD; Binding Sites; Boron Compounds; Chelating Agents; CHO Cells; Cricetinae; Europium; Fluorescein; Fluorescent Dyes; Humans; Interleukin-8; Ligands; Mutagenesis, Site-Directed; Receptors, Chemokine; Receptors, Interleukin; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Recombinant Proteins; Rhodamines; Spectrometry, Fluorescence