title: Complement regulatory activity of normal human intraocular fluid is mediated by MCP, DAF, and CD59.
abstract: OBJECTIVE
  To identify the molecules in normal human intraocular fluid (aqueous humor and vitreous) that inhibit the functional activity of the complement system.
  
  
  METHODS
  Aqueous humor and vitreous were obtained from patients with noninflammatory ocular disease at the time of surgery. Samples were incubated with normal human serum (NHS), and the mixture assayed for inhibition of the classical and alternative complement pathways using standard CH(50) and AH(50) hemolytic assays, respectively. Both aqueous humor and vitreous were fractionated by microconcentrators and size exclusion column chromatography. The inhibitory molecules were identified by immunoblotting as well as by studying the effect of depletion of membrane cofactor protein (MCP), decay-accelerating factor (DAF), and CD59 on inhibitory activity.
  
  
  RESULTS
  Both aqueous humor and vitreous inhibited the activity of the classical pathway (CH(50)). Microcentrifugation revealed the major inhibitory activity resided in the fraction with an M(r) >/= 3 kDa. Chromatography on an S-100-HR column demonstrated that the most potent inhibition was associated with the high-molecular-weight fractions (>/=19.5 kDa). In contrast to unfractionated aqueous and vitreous, fractions with an M(r) >/= 3 kDa also had an inhibitory effect on the alternative pathway activity (AH(50)). The complement regulatory activity in normal human intraocular fluid was partially blocked by monoclonal antibodies against MCP, DAF, and CD59. Immunoblot analysis confirmed the presence of these three molecules in normal intraocular fluid.
  
  
  CONCLUSIONS
  Our results demonstrate that normal human intraocular fluid (aqueous humor and vitreous) contains complement inhibitory factors. Furthermore, the high-molecular-weight factors appear to be the soluble forms of MCP, DAF, and CD59.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, Department of Medicine, Division of Cardiology, St. Louis University Medical Center, St. Louis, Missouri, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Sohn
authors.forename: H J
authors.initials: HJ
authors.lastname: Kaplan
authors.forename: H J
authors.initials: HJ
authors.lastname: Suk
authors.forename: P S
authors.initials: PS
authors.lastname: Bora
authors.forename: N S
authors.initials: NS
authors.lastname: Bora
mesh_terms: D015703:Antigens, CD / Q000378:metabolism*; D001082:Aqueous Humor / Q000502:physiology*; D018958:CD55 Antigens / Q000378:metabolism*; D018957:CD59 Antigens / Q000378:metabolism*; D002850:Chromatography, Gel; D015941:Complement Hemolytic Activity Assay; D003169:Complement Inactivator Proteins / Q000502:physiology*; D003170:Complement Pathway, Alternative / Q000502:physiology; D003171:Complement Pathway, Classical / Q000502:physiology; D003165:Complement System Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins / Q000378:metabolism*; D012164:Retinal Diseases / Q000378:metabolism; D014822:Vitreous Body / Q000502:physiology*
chemical_list: D015703:Antigens, CD; C497857:CD46 protein, human; D018958:CD55 Antigens; D018957:CD59 Antigens; D003169:Complement Inactivator Proteins; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins; D003165:Complement System Proteins