June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Inhibition of Innate Immune Response Alters Detoxification Capacity of ABCB1 Transporters in Trabecular Meshwork Cells
Author Affiliations & Notes
  • Algis Grybauskas
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL
  • Kevin Skuran
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL
  • Paulius Kuprys
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL
  • Beatrice Yue
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL
  • Paul Knepper
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL
    Ophthalmology, Northwestern Medical School, Chicago, IL
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 427. doi:
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      Algis Grybauskas, Kevin Skuran, Paulius Kuprys, Beatrice Yue, Paul Knepper; Inhibition of Innate Immune Response Alters Detoxification Capacity of ABCB1 Transporters in Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):427.

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

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Abstract

Purpose: The aqueous humor nourishes the avascular tissues of the anterior segment and the trabecular meshwork (TM) plays a role in removing xenobiotics. The innate immune system within the TM, particularly toll-like receptor 4 (TLR4) and its ligands, low molecular hyaluronic acid (LMW-HA) and lipopolysaccharide (LPS), play a significant role in maintaining a normal environment in the anterior chamber. We hypothesize that the innate immune system affects ATP-binding cassette sub-family member 1 (ABCB1, also known as p-glycoprotein and multidrug resistance protein 1) activity, altering the removal of toxic proteins, such as β-amyloid, heat shock proteins, and CD44.

Methods: Human TM cells and RAW 264.7 macrophages were plated onto 8-well chamber slides at 5000 cells/well overnight in 10% fetal bovine serum (FBS) cell growth medium. Two hours prior to treatment, the medium was changed to 0.1% FBS, and cells were challenged with 100 ng LMW-HA, (20 kDa), 100 ng high molecular weight hyaluronic acid (HMW-HA, 1000 kDa), 100 μM verapamil, 5 μM digoxin, 100 ng LPS, and/or 100 μM naloxone for 0.5, 1, 2, 4 h. Calcein-AM (0.25 μM, calcein-AM is cleaved by an esterase into a fluorescent product that is normally removed from the cell by ABCB1) was added for the final 15 min treatment. Cells were fixed at 4°C in 3% paraformaldehyde for 15 min, mounted with Vectashield with DAPI mounting medium, and analyzed with Leica confocal microscope and software.

Results: Following a 2h treatment, verapamil, an ABCB1 inhibitor, significantly (P<0.001) increased fluorescent calcein retention in the cytoplasm of both RAW 264.7 and TM cells compared to PBS control. Digoxin, an ABCB1 activator, increased calcein efflux (P<0.001). LPS significantly (P<0.001) reduced ABCB1 activity in TM cells. HMW-HA significantly (P<0.001) reduced ABCB1 activity, as did LMW-HA (P<0.02) and naloxone (P<0.001), a TLR4 inhibitor.

Conclusions: Both TLR4 ligands and inhibitors decrease ABCB1 activity, suggesting modulation of TLR4 is important in ABCB1 function. These results suggest that the innate immune inflammatory response plays a role in the ABCB1 detoxification pathway, and reducing inflammation may aid in the removal of toxic proteins in the TM.

Keywords: 615 neuroprotection • 555 immunomodulation/immunoregulation • 661 proteoglycans/glycosaminoglycans  
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