title: Effects of bilirubin on neutrophil responses in newborn infants.
abstract: BACKGROUND
  Newborns are susceptible to inflammatory diseases due to defects in clearing activated immune cells from tissues. Therefore, mechanisms have likely evolved to protect neonates from leukocyte-mediated cytotoxicity. Bilirubin has antioxidant activity, and it is possible that it also exerts effects on cellular immune responses in jaundiced infants.
  
  
  OBJECTIVE
  We hypothesize that bilirubin increases expression of antioxidant genes and decreases production of inflammatory proteins in neonatal neutrophils.
  
  
  METHODS
  Neutrophils were isolated from umbilical cord blood, and from adults for comparison, and treated with bilirubin (10-300 µmol/l, equivalent to unbound bilirubin 3-40 nmol/l), in the presence or absence of lipopolysaccharide (LPS). Expression of genes for antioxidant enzymes [superoxide dismutase (SOD), heme-oxygenase-1 (HO-1)] and heme-dependent enzymes involved in inflammation [NADPH oxidase-1 (NOX-1), cyclooxygenase-2 (COX-2)] was measured by PCR. Inflammatory cytokines were measured by bead array analysis using flow cytometry.
  
  
  RESULTS
  We found that LPS induced production of interleukin (IL)-8, IL-1β, and macrophage inhibitory protein-1β (MIP-1β). Bilirubin increased basal production of IL-8 and IL-1β, but downregulated LPS-induced generation of IL-8 and MIP-1β. It also upregulated SOD and HO-1 gene expression. We observed an unexpected bilirubin-induced increase in gene expression of NOX-1 in LPS-activated cells, and of COX-2 in both resting and activated cells.
  
  
  CONCLUSIONS
  These findings suggest that bilirubin suppresses inflammation and increases antioxidant enzyme generation in activated neonatal neutrophils. The unexpected increases in NOX-1 and COX-2 expression may represent an early response, with physiologic effects mitigated by increased antioxidant activity. Further studies will be needed to define levels of bilirubin that optimize its protective effects, while minimizing potential inflammatory toxicity.
authors.affiliation: Division of Neonatology, Department of Pediatrics, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ 08903, USA.
authors.forename: Barry
authors.initials: B
authors.lastname: Weinberger
authors.forename: Faith E
authors.initials: FE
authors.lastname: Archer
authors.forename: Suganya
authors.initials: S
authors.lastname: Kathiravan
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Hirsch
authors.forename: Alan M
authors.initials: AM
authors.lastname: Kleinfeld
authors.forename: Anna M
authors.initials: AM
authors.lastname: Vetrano
authors.forename: Thomas
authors.initials: T
authors.lastname: Hegyi
mesh_terms: D000975:Antioxidants; D001663:Bilirubin / Q000008:administration & dosage* / Q000032:analysis / Q000502:physiology; D017077:Culture Media, Conditioned / Q000737:chemistry; D051546:Cyclooxygenase 2 / Q000235:genetics; D016207:Cytokines / Q000096:biosynthesis; D005312:Fetal Blood / Q000166:cytology; D015870:Gene Expression / Q000187:drug effects; D051547:Heme Oxygenase-1 / Q000235:genetics; D006801:Humans; D051556:Hyperbilirubinemia, Neonatal; D007111:Immunity, Cellular / Q000187:drug effects; D007231:Infant, Newborn; D007249:Inflammation / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases / Q000235:genetics; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D013482:Superoxide Dismutase / Q000235:genetics
chemical_list: D000975:Antioxidants; D017077:Culture Media, Conditioned; D016207:Cytokines; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D051547:Heme Oxygenase-1; D051546:Cyclooxygenase 2; D013482:Superoxide Dismutase; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C402799:NOX1 protein, human; D001663:Bilirubin