March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Oxidative Stress Sensitizes RPE Cells To Complement-Mediated Injury In A Lectin Pathway- And Malondialdehyde Epitope-Dependent Manner
Author Affiliations & Notes
  • Baerbel Rohrer
    Ophthalmology, Med Univ of South Carolina, Charleston, South Carolina
  • Kusumam Joseph
    Ophthalmology, Med Univ of South Carolina, Charleston, South Carolina
  • Liudmila Kulik
    Medicine, University of Colorado School of Medicine, Aurora, Colorado
  • Steffen Thiel
    Biomedicine, Aarhus University, Aarhus, Denmark
  • Nicole Thielens
    Institute for Structural Biology, Grenoble, France
  • V M. Holers
    Medicine, University of Colorado School of Medicine, Aurora, Colorado
  • Footnotes
    Commercial Relationships  Baerbel Rohrer, None; Kusumam Joseph, None; Liudmila Kulik, None; Steffen Thiel, None; Nicole Thielens, None; V. M. Holers, None
  • Footnotes
    Support  NIH grant EY019320, a Department for Veterans Affairs merit award RX000444, Foundation Fighting Blindness and Research to Prevent Blindness.
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 2717. doi:
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    • Get Citation

      Baerbel Rohrer, Kusumam Joseph, Liudmila Kulik, Steffen Thiel, Nicole Thielens, V M. Holers; Oxidative Stress Sensitizes RPE Cells To Complement-Mediated Injury In A Lectin Pathway- And Malondialdehyde Epitope-Dependent Manner. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2717.

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      © ARVO (1962-2015); The Authors (2016-present)

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  • Supplements
Abstract

Purpose: : Uncontrolled activation of the alternative complement pathway is thought to be associated with age-related macular degeneration (AMD). Previously, we have shown that in retinal pigmented epithelium (RPE) monolayers, oxidative stress reduced complement inhibitor expression and function on the cell surface, resulting in sublytic activation of the membrane attack complex . Here we examined the potential ligand and pathway(s) involved in initiating complement-dependent RPE cell damage by oxidative stress.

Methods: : ARPE-19 cells were grown as monolayers on transwell plates. Sublytic complement activation was induced by challenging monolayers with H2O2 in the presence of complement-sufficient normal human serum (NHS). Since sublytic complement activation results in VEGF release, which in turn reduces barrier function, transepithelial electrical resistance (TER) measurements were used as a measure of cell injury.

Results: : (1) TER deteriorated rapidly in H2O2-exposed monolayers upon adding NHS as a source of complement. While the effect required alternative pathway (AP) activation, the AP was not sufficient, since elimination of MBL/MASP prevented TER reduction. Elimination of C1q from NHS had no effect. (2) Reconstitution experiments revealed that both ficolin/MASP and MBL/MASP can activate the complement cascade. (3) Ig-depleted human serum or mouse serum from RAG-deficient mice eliminated the effect, demonstrating a requirement for Ig to initiate injury. (4) TER reduction could be reinstituted using natural IgM-producing monoclonal antibodies (mAbs), with the most effective IgM recognizing a malondialdehyde (MDA) epitope.

Conclusions: : Oxidative stress sensitizes RPE cells to the deleterious effects of a dysregulated complement system. Using a combination of complement-depletion and reconstitution strategies, we have shown that the lectin pathway (LP) is required to initiate the complement cascade, which is then further amplified by the AP. LP activation is potentially triggered by natural IgM bound to MDA. In summary, we have developed a model that reconstitutes complement activation in oxidatively stressed RPE, linking together important molecular events involved in AMD, including the presence of autoantibodies to MDA-related epitopes that are newly expressed.

Keywords: age-related macular degeneration • retinal pigment epithelium • immunomodulation/immunoregulation 
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