June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Human lipofuscin induces a distinct immune reaction and elevated VEGF levels in microglial cells in vitro
Author Affiliations & Notes
  • Martin Dominik Leclaire
    Research Laboratory of the Department of Ophthalmology, University of Muenster Medical Center, Muenster, Germany
  • Peter Heiduschka
    Research Laboratory of the Department of Ophthalmology, University of Muenster Medical Center, Muenster, Germany
  • Gerburg Nettels-Hackert
    Research Laboratory of the Department of Ophthalmology, University of Muenster Medical Center, Muenster, Germany
  • Jeanette Koenig
    German Institute of Human Nutrition, Potsdam-Rehbruecke, Germany
  • Tilman Grune
    German Institute of Human Nutrition, Potsdam-Rehbruecke, Germany
  • Constantin Uhlig
    Cornea Bank of the Department of Ophthalmology, University of Muenster Medical Center, Muenster, Germany
  • Uwe Hansen
    Institute of Experimental Musculoskeletal Medicine, Faculty of Medicine, University of Muenster, Muenster, Germany
  • Nicole Eter
    Research Laboratory of the Department of Ophthalmology, University of Muenster Medical Center, Muenster, Germany
  • Footnotes
    Commercial Relationships   Martin Dominik Leclaire, None; Peter Heiduschka, None; Gerburg Nettels-Hackert, None; Jeanette Koenig, None; Tilman Grune, None; Constantin Uhlig, None; Uwe Hansen, None; Nicole Eter, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 5376. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Martin Dominik Leclaire, Peter Heiduschka, Gerburg Nettels-Hackert, Jeanette Koenig, Tilman Grune, Constantin Uhlig, Uwe Hansen, Nicole Eter; Human lipofuscin induces a distinct immune reaction and elevated VEGF levels in microglial cells in vitro. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5376.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : Uptake of lipofuscin (LF) in microglial cells has been reported in subretinal microglia in older mice, which suggests a link to AMD pathology where microglia also accumulate in the subretinal space. The detailed reaction of the microglia towards LF is not known yet. We therefore studied effects of LF on microglial cells in vitro.
.

Methods : Human LF was extracted from human donor eyes. Immortalized BV-2 microglial cells and primary cells (PC) from murine CNS were incubated with a mixture of medium and lipofuscin in different concentrations for 24 hours. Further samples were additionally treated with hydrocortisone, minocycline and the tripeptide TKP. We analyzed the phagocytosis of LF by detecting its autofluorescence. Immunohistochemical stainings for microglial surface markers (CD-11b, F-4/80 and Iba-1) and also for VEGF were performed. In order to analyze cytokine release, we used a multiplex immunoassay for 17 pro-inflammatory cytokines and also an ELISA for VEGF.

Results : Apart from morphological changes in the microglial cells (all CD-11b+, F-4/80+, Iba-1+) treated with LF, autofluorescence signals from inside these cells demonstratedphagocytosis of the LF. Immunohistochemical staining revealed elevated VEGF expression in BV-2 cells treated with LF. Highly elevated levels of pro-inflammatory cytokines in the supernatants for both, BV-2 cells and primary cells, were found by the multiplex assay, in particular IL-6, IL-1α and TNF-α (Fig. 1). Highly elevated VEGF levels in the supernatants of BV-2 cells after LF stimulation correlating with the LF concentration were found by ELISA (Fig. 2). However, in our primary cell culture, VEGF-levels were not significantly elevated (p=0.06).
Hydrocortisone significantly inhibited the immune reaction suppressing, among others, the cytokines that had the greatest increase after the exposure to LF (i.a. IL-6, IL-1 and TNF-α; all at least p<0.05). TKP and minocycline suppressed microglial proliferation but had less impact on cytokine levels.

Conclusions : We were able to demonstrate for the first time that microglial cells phagocytose fast and completely human LF in vitro. Our results demonstrate the inflammatory potency of the microglia upon interaction with LF and hence, point to their potential role in degeneration. Elevated VEGF levels more specifically present a link to neovascularization in AMD.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

 

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×