title: Combined inhibition of complement and CD14 abolish E. coli-induced cytokine-, chemokine- and growth factor-synthesis in human whole blood.
abstract: The relative role of complement and CD14 in E. coli-induced cytokine synthesis in an in vitro human whole blood model of sepsis was examined. Fresh lepirudin-anticoagulated whole blood was incubated with E. coli for 2h. Monoclonal antibodies or a C5a receptor antagonist were used to block complement. Inflammatory mediators (n=27) were measured by multiplex technology, selected cytokine mRNA by real time PCR, and CD11b, oxidative burst and phagocytosis by flow cytometry. E. coli significantly increased 18 of the 27 inflammatory mediators, including proinflammatory cytokines (TNF-alpha, IL-6, INF-gamma and IL-1beta), chemokines (IL-8, MCP-1, MIP-1alpha, MIP-1beta, eotaxin and IP-10), growth factors (VEGF, FGF-basic, G-CSF and GM-CSF) and other interleukins (IL-9, IL-15 and IL-17). Notably, the increases in all mediators were abolished by a combined inhibition of CD14 and complement using anti-C2 and anti-factor D in combination, whereas the relative effect of the inhibition of complement and CD14 varied. In comparison, a C5a receptor antagonist and anti-CD14 in combination reduced cytokine synthesis less efficiently. Real time PCR analysis confirmed that the cytokine synthesis was blocked at the mRNA level. Similarly, E. coli-induced CD11b up-regulation, oxidative burst and phagocytosis was totally inhibited by CD14, anti-C2 and anti-factor D in combination after 2h incubation. In conclusion, the combined inhibition of complement using anti-C2, anti-factor D and CD14 almost completely inhibits the E. coli-induced inflammatory response. The combined approach may therefore be a new treatment regimen in Gram-negative sepsis.
authors.affiliation: Department of Laboratory Medicine, Nordland Hospital, Bodø, Norway. Ole.Lars.Brekke@nlsh.no
authors.forename: Ole-Lars
authors.initials: OL
authors.lastname: Brekke
authors.forename: Dorte
authors.initials: D
authors.lastname: Christiansen
authors.forename: Hilde
authors.initials: H
authors.lastname: Fure
authors.forename: Anne
authors.initials: A
authors.lastname: Pharo
authors.forename: Michael
authors.initials: M
authors.lastname: Fung
authors.forename: Johan
authors.initials: J
authors.lastname: Riesenfeld
authors.forename: Tom Eirik
authors.initials: TE
authors.lastname: Mollnes
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D018925:Chemokines / Q000096:biosynthesis* / Q000097:blood; D003165:Complement System Proteins / Q000276:immunology*; D016207:Cytokines / Q000096:biosynthesis* / Q000097:blood; D004926:Escherichia coli / Q000276:immunology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000097:blood; D018950:Lipopolysaccharide Receptors / Q000276:immunology*; D012333:RNA, Messenger / Q000276:immunology; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000037:antagonists & inhibitors / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; C488033:C5AR1 protein, human; D018925:Chemokines; D016207:Cytokines; D036341:Intercellular Signaling Peptides and Proteins; D018950:Lipopolysaccharide Receptors; D012333:RNA, Messenger; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D003165:Complement System Proteins