title: Transient exposure to tumor necrosis factor-alpha inhibits collagen accumulation by cultured hypertrophic scar fibroblasts.
abstract: BACKGROUND
  Hypertrophic scars (HS) are frequent consequences of deep dermal injury, such as deep partial-thickness burns and abrasions, and are characterized by overproduction of collagen. In vitro studies have shown that cultured HS fibroblasts produce elevated levels of collagen and insulin-like growth factor-binding protein 3 (IGFBP-3). Additionally, histological studies have indicated HS contain fewer tumor necrosis factor alpha (TNF-alpha)-positive infiltrating cells and express lower levels of TNF-alpha mRNA, suggesting TNF-alpha, which can inhibit collagen expression in some systems, may function to deactivate the wound healing process in scars. HS also exhibit increased levels of transforming growth factor beta (TGF-beta), a factor that stimulates collagen and extracellular matrix deposition by fibroblasts and also stimulates IGFBP-3 expression. In some systems, IGFBP-3 mediates the effects of TGF-beta. The present study sought to determine the effects of continuous and transient TNF-alpha exposure on collagen and IGFBP-3 expression by cultured HS fibroblasts and to investigate the role of IGFBP-3 in collagen accretion by HS fibroblasts.
  
  
  METHODS
  Superficial and deep dermal HS fibroblasts from four patients were cultured. Fibroblasts were cultured in serum-free medium and exposed to 0-2 ng/ml TNF-alpha for 0, 1, 4, or 72 h. After 72 h of culture, medium samples were processed for Western blot analysis of type I collagen accumulation or for ligand blot analysis of IGFBP-3 accumulation. The effects of an anti-IGFBP-3 neutralizing antibody on collagen accumulation were also assessed.
  
  
  RESULTS
  Treatment of superficial and deep HS fibroblasts with TNF-alpha resulted in dose-dependent decreases in accumulation of both type I collagen and IGFBP-3 in the culture medium (P < 0.01). However, using the anti-IGFBP-3 neutralizing antibody, a causal relationship between decreased IGFBP-3 and decreased collagen accumulation could not be demonstrated. Transient exposure of cultured HS fibroblasts to TNF-alpha for as little as 1 h was as effective as continuous exposure to TNF-alpha for 72 h in inhibiting collagen accumulation.
  
  
  CONCLUSIONS
  These results support the hypothesis that TNF-alpha functions as a wound healing deactivation signal that is deficient in HS. Although TNF-alpha inhibited accretion of both collagen and IGFBP-3, the role of IGFBP-3 in HS remains unresolved. This study suggests that transient TNF-alpha exposure may be used to inhibit collagen overaccumulation in HS and that the timing of TNF-alpha exposure following dermal injury may not be critical for this inhibition.
authors.affiliation: Department of Medicine, University of Washington, Seattle, Washington 98195, USA.
authors.forename: V
authors.initials: V
authors.lastname: Kitzis
authors.forename: L H
authors.initials: LH
authors.lastname: Engrav
authors.forename: L S
authors.initials: LS
authors.lastname: Quinn
mesh_terms: D000328:Adult; D002478:Cells, Cultured; D002921:Cicatrix / Q000378:metabolism*; D003094:Collagen / Q000096:biosynthesis*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D006984:Hypertrophy; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000502:physiology; D008233:Lymphotoxin-alpha / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D018972:Insulin-Like Growth Factor Binding Protein 3; D008233:Lymphotoxin-alpha; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D003094:Collagen