Modulation of endotoxin-induced endothelial activity by microtubule depolymerization.
BACKGROUND
Endotoxin not only activates the Toll-mediated signaling pathway within endothelial cells that leads to neutrophil migration but also causes the polymerization of microtubules. The potential role of this polymerization event, however, is unknown.


METHODS
Human umbilical vein endothelial cells stimulated with endotoxin were pretreated with or without the microtubule depolymerizing agent colchicine. Toll-mediated signaling events and protein production were in turn investigated by Western blot, gel shift, and enzyme-linked immunosorbent assay. Finally, neutrophil adhesion was assayed fluorometrically under the various conditions.


RESULTS
Endotoxin led to activation of the various Toll-mediated pathways, production of intercellular adhesion molecule-1 and interleukin-8, and subsequent neutrophil adhesion. Pretreatment with colchicine led to selective inhibition of anti-dual phosphorylated extracellular signal-regulated kinase-1/2, anti-dual phosphorylated c-jun N-terminal kinase, and adaptor protein-1; selective enhancement of p38; and no effect on nuclear factor-kappaB. This selective modulation of intracellular signaling resulted in attenuated intercellular adhesion molecule-1, interleukin-8 and prostaglandin E2 production, but enhanced cyclooxygenase-2 expression. As a result, microtubule disruption led to a significant reduction in neutrophil adhesion.


CONCLUSIONS
Microtubule formation is essential to optimal endotoxin-induced intracellular signaling through anti-dual phosphorylated extracellular signal-regulated kinase-1/2, anti-dual phosphorylated c-jun N-terminal kinase, and adaptor protein-1. Failure of these signaling events is associated with a marked reduction in the formation of a proadhesive phenotype that may prove to be beneficial in modulating neutrophil recruitment during sepsis.
Cuschieri
Joseph
J
Department of Surgery, Division of Trauma/Critical Care, University of Cincinnati, 231 Albert Sabin Way, ML 558, Cincinnati, OH 45267-0558, USA. cush98@aol.com
Gourlay
David
D
Garcia
Iris
I
Jelacic
Sandra
S
Maier
Ronald V
RV
D033961:Adaptor Protein Complex 1 / Q000276:immunology; D015153:Blotting, Western; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000276:immunology; D029721:Drosophila Proteins; D024202:Electrophoretic Mobility Shift Assay; D004730:Endothelium, Vascular / Q000166:cytology* / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000276:immunology; D016209:Interleukin-8 / Q000276:immunology; D007527:Isoenzymes / Q000276:immunology; D048031:JNK Mitogen-Activated Protein Kinases; D008070:Lipopolysaccharides / Q000009:adverse effects*; D008562:Membrane Glycoproteins / Q000276:immunology; D008565:Membrane Proteins; D008870:Microtubules / Q000276:immunology*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000276:immunology; D016328:NF-kappa B / Q000276:immunology; D018375:Neutrophil Activation / Q000276:immunology; D020556:Neutrophil Infiltration / Q000276:immunology; D011108:Polymers; D011451:Prostaglandin-Endoperoxide Synthases / Q000276:immunology; D011956:Receptors, Cell Surface / Q000276:immunology; D018805:Sepsis / Q000276:immunology*; D015398:Signal Transduction / Q000276:immunology; D051193:Toll-Like Receptors; D014471:Umbilical Veins / Q000166:cytology
D033961:Adaptor Protein Complex 1; D029721:Drosophila Proteins; D016209:Interleukin-8; D007527:Isoenzymes; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D016328:NF-kappa B; D011108:Polymers; D011956:Receptors, Cell Surface; D051193:Toll-Like Receptors; D018799:Intercellular Adhesion Molecule-1; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D048031:JNK Mitogen-Activated Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D015232:Dinoprostone; D003078:Colchicine