ABSTRACT
Previous animal studies have demonstrated that following systemic administration phosphorothioate oligodeoxynucleotides (S-ODNs) are primarily excreted by the kidneys and that renal tissue levels of S-ODNs exceed that of other organs. Thus, the kidney may be an ideal target organ for application of antisense S-ODNs in vivo. We examined which cells within the rat kidney have uptake of radiolabeled S-ODNs following intravenous infusion. A 20-base 35S-ODN was infused into 6 adult male Wistar rats. Three animals each were sacrificed 30 min and 4 h after infusion. The kidneys were then removed, fixed, and tissue autoradiography was performed. Similar results were obtained in both groups. The highest level of radioactivity was seen within the proximal tubules. Lower levels of activity were seen within the glomerulus, the parietal epithelial cells of Bowman's space, and distal tubular cells. Very weak activity was also detected within the cells of the loop of Henle and the medullary collecting ducts. These results demonstrated that within the kidney S-ODNs were taken up primarily by proximal tubular cells, with much lower uptake by cells in other segments of the nephron.
Subject(s)
Kidney/metabolism , Oligonucleotides, Antisense/pharmacokinetics , Thionucleotides/pharmacokinetics , Animals , Autoradiography , Infusions, Intravenous , Kidney/cytology , Kidney Tubules/cytology , Kidney Tubules/metabolism , Male , Oligonucleotides, Antisense/administration & dosage , Rats , Rats, Wistar , Sulfur Radioisotopes , Thionucleotides/administration & dosageABSTRACT
In this report, we studied the effects of transforming growth factor (TGF)-beta 1 on lipopolysaccharide (LPS)-induced expression of endothelial cell (EC) adhesion molecules. Confluent human umbilical cord vein EC cultures were stimulated with Escherichia coli LPS and TGF-beta 1, alone or in combination for various times and evaluated for expression of ICAM-1, E-selectin, and VCAM-1 by immunofluorescence and radioimmunoassay. Effects of LPS and/or TGF-beta 1 on cell growth were also studied by 3H-thymidine incorporation. Both LPS and TGF-beta 1 alone stimulated EC expression of the adhesion molecules in a dose-dependent manner. The effects of TGF-beta 1 on LPS induction of the adhesion molecules varied with LPS concentration and treatment time, mode, and duration. Pretreatment with TGF-beta 1 for 24 h greatly augmented LPS induction of ICAM-1 and VCAM-1 expression, but decreased E-selectin expression. TGF-beta 1 also enhanced expression of the adhesion molecules in cells that were pretreated with 1 microgram/mL LPS for 60 min. Concomitant treatment with TGF-beta 1/LPS resulted in significant increases in ICAM-1 but decreases in VCAM-1 expression. TGF-beta 1 effects on LPS induction of the adhesion molecules were more prominent at lower LPS levels (.001, .01 microgram/mL). Both LPS and TGF-beta 1 suppressed thymidine incorporation in a dose-related pattern. These data suggest that TGF-beta 1 has additive and antagonistic effects on LPS induction of the adhesion molecules and that the cell responsiveness to the stimuli in the expression is related to growth condition of the cells. In conclusion, our findings suggest that TGF-beta 1 exhibits pro-inflammatory and anti-inflammatory activities in human endothelial cells and may play an important role in regulating leukocyte adherence and extravasation under LPS-induced inflammatory conditions.
Subject(s)
E-Selectin/biosynthesis , Endothelium, Vascular/drug effects , Gene Expression Regulation/drug effects , Intercellular Adhesion Molecule-1/biosynthesis , Lipopolysaccharides/pharmacology , Transforming Growth Factor beta/pharmacology , Vascular Cell Adhesion Molecule-1/biosynthesis , Cells, Cultured , DNA Replication/drug effects , Drug Interactions , E-Selectin/genetics , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Fluorescent Antibody Technique, Indirect , Humans , Intercellular Adhesion Molecule-1/genetics , Vascular Cell Adhesion Molecule-1/geneticsABSTRACT
Endotoxin (lipopolysaccharide, LPS) is known to induce inflammatory responses, such as monocyte/macrophage adherence, migration, and accumulation. Recruitment and accumulation of macrophages during infection and inflammation are regulated by integrin-mediated cell-extracellular matrix interactions. In the present report, we studied the effects of LPS on the expression of VLA-5 (alpha 5 beta 1), VLA-3 (alpha 3 beta 1), and VLA-2 (alpha 2 beta 1) integrins and fibronectin (FN) by human alveolar macrophages in an attempt to understand the mechanism by which LPS regulates macrophage adhesion to matrix proteins. Bronchoalveolar lavage macrophages were treated with varying concentrations of Escherichia coli LPS for different times and evaluated for expression of the integrins and FN by immunofluorescence, immunoelectron microscopy, autoradiography, and radioimmunoassay. Immunofluorescent and immunoelectron microscopic observations showed that VLA integrins were constitutively expressed on the cell surface and concentrated on the microvilli and pseudopodia of the macrophages. The effects of LPS on expression of the integrins were dose and time related. VLA-5 expression was increased after 30 min of stimulation by LPS, suggesting that LPS may induce rapid secretion of the integrin. However, incubations with LPS longer than 30 min decreased VLA-5 expression in a dose-dependent pattern. LPS also caused dose-related decreases in the expression of VLA-3 and VLA-2 integrins and increases of intracellular FN 24 h after stimulation. The results suggest that a prolonged exposure to LPS may impede VLA integrin-mediated migration and result in local accumulation of macrophages in the lung.
