Endothelial cell activation by leukocyte microparticles.
The ability of polymorphonuclear leukocytes (PMNs) to modulate endothelial cell (EC) activation was investigated. Adding PMNs to cultured HUVECs resulted in a release of IL-6 (888 +/- 71 pg/ml, a 35-fold increase over release by the two cell types alone) and IL-8 (45.2 +/- 14.5 ng/ml, a 6.4-fold over PMN release alone and a 173-fold increase over EC release alone). In contrast, the release of TNF-alpha, IL-1beta, and platelet-derived growth factor was not affected by the EC-PMN coculture. Neutralizing mAbs to ICAM-1 or beta2 integrins or a physical segregation of PMNs and ECs did not reduce EC stimulation. In contrast, cell-free supernatants of PMNs recapitulated EC activation with an 18-fold up-regulation of EC IL-6 mRNA. The filtration of PMN supernatant or PMN pretreatment with metabolic antagonists or membrane cross-linking agents all suppressed EC activation. By flow cytometry, PMNs released in the supernatant, heterogeneous membrane-derived microparticles containing discrete proteins of 28 to 250 kDa as resolved by SDS-PAGE. PMN microparticle formation was enhanced by inflammatory stimuli, including formyl peptide and phorbol ester, and was time-dependent, reaching a plateau after a 1-h incubation from stimulation. Purified PMN microparticles induced EC IL-6 release in a reaction that was quantitatively indistinguishable from that observed with unfractionated PMN supernatant and unaffected by a neutralizing Ab to soluble IL-6R. These findings demonstrate that membrane microparticles released from stimulated PMNs are competent inflammatory mediators to produce EC activation and cytokine gene induction.
Department of Pathology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536, USA.
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D002450:Cell Communication / Q000276:immunology*; D002478:Cells, Cultured; D018920:Coculture Techniques; D003594:Cytoplasmic Granules / Q000276:immunology; D004730:Endothelium, Vascular / Q000166:cytology / Q000276:immunology*; D006801:Humans; D015850:Interleukin-6 / Q000276:immunology; D009504:Neutrophils / Q000166:cytology / Q000276:immunology*
D015850:Interleukin-6