title: Synergistic induction of IL-10 by hypertonic saline solution and lipopolysaccharides in murine peritoneal macrophages.
abstract: BACKGROUND
  Liver injury after ischemia/reperfusion is an important cause of morbidity in surgical patients. We have shown that the preconditioning of animals that were subjected to liver ischemia/reperfusion with hypertonic saline solution (HTS) prevented injury by inhibiting Kupffer cell tumor necrosis factor (TNF) production. We postulated that the induction of anti-inflammatory interleukin-10 (IL-10) by HTS might contribute to protection.
  
  
  METHODS
  Murine thioglycolate--elicited peritoneal exudative macrophages (PEMs) were used to model the effects of HTS on IL-10 release from Kupffer cells. Cells were preconditioned with 500 mOsm HTS (or isotonic saline medium) for 2 hours and then stimulated with lipopolysaccharide (LPS; 1 microg/mL) or vehicle for 4 hours under isotonic conditions. TNF-alpha and IL-10 were measured in the culture supernatant by enzyme-linked immunosorbent assay; TNF, IL-10, and SOCS-3 messenger RNA expression were assessed by Northern blot. NF-kappa B activation was examined by electrophoretic mobility shift assay and Western blot for I kappa B degradation.
  
  
  RESULTS
  In the absence of LPS, isotonic medium--and HTS-pretreated PEMs produced little IL-10 (24.9 +/- 66.0 and 0 pg/mL, respectively); however, stimulation of PEMs with LPS increased IL-10 (134.9 +/- 72.2 pg/mL). Preconditioning with HTS significantly augmented LPS-induced IL-10 production, resulting in a 2-fold increase in IL-10 compared with the isotonic solution LPS group (270.7 +/- 106.8 pg/mL; P <.01). HTS alone increased IL-10 mRNA levels and markedly augmented levels induced by LPS alone. To determine whether IL-10 accounted for HTS-induced TNF inhibition, cells from IL-10 knockout animals were studied. A lack of IL-10 did not reverse the inhibitory effect of HTS on LPS-induced TNF. NF-kappa B activation was the same in HTS-and isotonic solution--pretreated groups after LPS.
  
  
  CONCLUSIONS
  HTS augments IL-10 induction by LPS at the gene level. Although TNF is reduced, it is not causally related to increased IL-10 or altered NF-kappa B signaling. HTS might exert its beneficial effects by independently modulating pro- and anti-inflammatory molecules, accounting for the potent immunomodulation exerted by HTS in vivo.
authors.affiliation: Department of Surgery, University Health Network, Toronto, Ontario, Canada.
authors.forename: G D
authors.initials: GD
authors.lastname: Oreopoulos
authors.forename: S
authors.initials: S
authors.lastname: Bradwell
authors.forename: Z
authors.initials: Z
authors.lastname: Lu
authors.forename: J
authors.initials: J
authors.lastname: Fan
authors.forename: R
authors.initials: R
authors.lastname: Khadaroo
authors.forename: J C
authors.initials: JC
authors.lastname: Marshall
authors.forename: Y H
authors.initials: YH
authors.lastname: Li
authors.forename: O D
authors.initials: OD
authors.lastname: Rotstein
mesh_terms: D000818:Animals; D004357:Drug Synergism; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D016753:Interleukin-10 / Q000032:analysis / Q000235:genetics*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000378:metabolism; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D012097:Repressor Proteins; D012462:Saline Solution, Hypertonic / Q000494:pharmacology*; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000235:genetics
chemical_list: D008070:Lipopolysaccharides; D016328:NF-kappa B; D011506:Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; D012462:Saline Solution, Hypertonic; C491411:Socs3 protein, mouse; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10