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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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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.
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.
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.
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.
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