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Department of Pathology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536
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-
, IL-1ß, and platelet-derived growth factor was not
affected by the EC-PMN coculture. Neutralizing mAbs to ICAM-1 or
ß2 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.
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