title: Biologically active fibronectin fragments stimulate release of MCP-1 and catabolic cytokines from murine retinal pigment epithelium.
abstract: OBJECTIVE
  High-temperature requirement serine protease (HTRA1) was identified as a candidate age-related macular degeneration gene in multiple genetic studies in humans. To date, no functional studies have shown a mechanism for HTRA1 to instigate ocular tissue abnormalities. In the present study, the authors focused on a substrate of HTRA1, fibronectin, because fibronectin fragments (Fnfs) stimulate biochemical events in other age-related degenerative diseases that are analogous to changes associated with age-related macular degeneration (AMD). The purpose of the study was to determine whether Fnfs stimulate the release of proinflammatory and catabolic cytokines from murine retinal pigment epithelium (RPE).
  
  
  METHODS
  Fibronectin was purified from murine serum by gelatin cross-linked agarose chromatography and subsequently was enzymatically digested with alpha-chymotrypsin. The bioactivity of Fnfs was verified by measuring levels of IL-6 and TNF-alpha in Fnf-exposed murine splenocytes. To analyze the effect of Fnfs on RPE, cytokine and chemokine levels in RPE culture supernatants were assayed by ELISA.
  
  
  RESULTS
  IL-6 and TNF-alpha proinflammatory cytokines were released from primary murine splenocytes in proportion to the dose and length of Fnf treatment, indicating that alpha-chymotryptic digests of fibronectin are biologically active. Fnf treatment of murine RPE cells stimulated the release of microgram and nanogram levels of IL-6, MMP-3, MMP-9, and MCP-1, whereas only picogram levels were detected in untreated cells.
  
  
  CONCLUSIONS
  Fnfs stimulate the release of proinflammatory cytokines, matrix metalloproteinases, and monocyte chemoattractant protein from murine RPE cells. This observation indicated that Fnfs could contribute to ocular abnormalities by promoting inflammation, catabolism, and monocyte chemoattraction.
authors.affiliation: National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-1857, USA. drbob@nei.nih.gov
authors.forename: Bobbie Ann
authors.initials: BA
authors.lastname: Austin
authors.forename: Baoying
authors.initials: B
authors.lastname: Liu
authors.forename: Zhuqing
authors.initials: Z
authors.lastname: Li
authors.forename: Robert B
authors.initials: RB
authors.lastname: Nussenblatt
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism*; D018925:Chemokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins / Q000494:pharmacology*; D015850:Interleukin-6 / Q000378:metabolism*; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000494:pharmacology*; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D013997:Time Factors
chemical_list: C491768:Ccl2 protein, mouse; D018932:Chemokine CCL2; D018925:Chemokines; D005353:Fibronectins; D015850:Interleukin-6; D010446:Peptide Fragments; D019278:Matrix Metalloproteinase 3; D020780:Matrix Metalloproteinase 9