title: Identification of meningococcal LPS as a major monocyte activator in IL-10 depleted shock plasmas and CSF by blocking the CD14-TLR4 receptor complex.
abstract: We have examined the in vitro stimulatory effects of lipopolysaccharide (LPS)-containing samples (meningococcal shock plasma, n = 10; non-shock plasma, n = 10; cerebrospinal fluid (CSF), n = 7) before and after immunodepletion of interleukin (IL)-10 in a monocyte target assay. We also studied the stimulatory effects of plasma collected from 3 patients with lethal septicemia caused by Streptococcus pneumoniae without detectable LPS but with 100-fold increased levels of heat-shock protein 70 (HSP70). HSP70 may, like LPS, activate monocytes via the Toll-like receptor 4 (TLR4). The samples were analyzed for LPS, tumor necrosis factor (TNF)-alpha, IL-10 and HSP70; applied on human monocytes (purity > 95%) before and after IL-10 immunodepletion, in the absence or presence of CD14 blocking mAb (60bca) or the lipid A antagonist, Rhodobacter sphaeroides diphosphoryl lipid A (RsDPLA) which blocks TLR4. Monocyte activation was measured by increased TNF-alpha secretion and tissue factor (TF) up-regulation by monocyte procoagulant activity (PCA). There was a positive correlation between patient plasma LPS levels (n = 10) and increases in TNF-alpha secretion by the monocytes after immunodepletion of IL-10 (r = 0.82). Pretreatment of the monocytes with mAbCD14 or RsDPLA reduced TNF-alpha secretion to median 5% and 12%, respectively, of the levels before the receptor complex was blocked. The median levels of HSP70 were 543 ng/ml (range, 468-962 ng/ml) in pneumococcal shock plasma, 81.5 ng/ml (range, 41-331 ng/ml) in meningococcal shock plasma and 24 ng/ml (range, < 0.8-41 ng/ml) in meningococcal non-shock plasma. Pneumococcal septic shock plasmas with significantly higher levels of HSP70 (P < 0.05) did not induce TNF-alpha secretion in the monocytes. The results strongly suggest that LPS in meningococcal shock plasma is the major activator of monocytes whereas HSP70 (in plasma concentrations up to 963 ng/ml) does not activate monocytes in this assay.
authors.affiliation: Department of Pediatrics, Ullevål University Hospital, 0407 Oslo, Norway. a.k.bjerre@ioks.uio.no
authors.forename: Anna
authors.initials: A
authors.lastname: Bjerre
authors.forename: Berit
authors.initials: B
authors.lastname: Brusletto
authors.forename: Reidun
authors.initials: R
authors.lastname: Øvstebø
authors.forename: Gun Britt
authors.initials: GB
authors.lastname: Joø
authors.forename: Peter
authors.initials: P
authors.lastname: Kierulf
authors.forename: Petter
authors.initials: P
authors.lastname: Brandtzaeg
mesh_terms: D000293:Adolescent; D000328:Adult; D019138:Antibodies, Blocking / Q000494:pharmacology; D002648:Child; D002675:Child, Preschool; D018840:HSP70 Heat-Shock Proteins / Q000097:blood / Q000494:pharmacology; D006801:Humans; D007223:Infant; D016753:Interleukin-10 / Q000097:blood / Q000172:deficiency; D018950:Lipopolysaccharide Receptors / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000097:blood* / Q000633:toxicity; D008262:Macrophage Activation / Q000187:drug effects / Q000502:physiology; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008589:Meningococcal Infections / Q000097:blood / Q000503:physiopathology; D008875:Middle Aged; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism*; D009345:Neisseria meningitidis / Q000737:chemistry / Q000472:pathogenicity; D011135:Polysaccharides, Bacterial; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018805:Sepsis / Q000097:blood / Q000503:physiopathology; D012772:Shock, Septic / Q000097:blood / Q000134:cerebrospinal fluid; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D019138:Antibodies, Blocking; D018840:HSP70 Heat-Shock Proteins; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D011135:Polysaccharides, Bacterial; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10