ABCB1 transporter and Toll-like receptor 4 in trabecular meshwork cells

PURPOSE: The aqueous humor nourishes the avascular tissues of the anterior segment, and the trabecular meshwork (TM) plays a role in the efflux of endogenous substances and xenobiotics from the aqueous humor. ATP (ATP)-binding cassette (ABC) transporter superfamily members respond to stressors such as hypoxia, cytokine signaling, and aging. The innate immune system within the TM, particularly Toll-like receptor 4 (TLR4) and its ligands, e.g., low-molecular-weight hyaluronic acid (LMW-HA) and lipopolysaccharide (LPS), plays a significant role in maintaining a normal environment in the anterior chamber. We hypothesize that the innate immune system may interact with ATP-binding cassette sub-family members ABCB1 (p-glycoprotein and multidrug resistance protein 1) to detoxify xenobiotics from the aqueous humor and in the TM. METHODS: Cell lysates of human TM cells, RAW 264.7 macrophages, and PC12 cells were subjected to western blot analysis. The TM cells were positive for TLR4, ABCB1, and CYP3A5 and were negative for the ABCC1 transporter. Human TM cells and RAW 264.7 macrophages were plated on eight-well chamber slides at 5,000 cells/well overnight in 10% fetal bovine serum (FBS) cell growth medium. The medium was changed to 0.1% FBS 2 h before treatment. Cells were challenged with 1 and 10 mM lactate, 100 ng LMW-HA (20 kDa), 100 ng high-molecular-weight HA (HMW-HA, 1,000 kDa), 100 ng LPS, and/or 100 μM naloxone for 0.5, 1, 2, and 4 h. Calcein acetyoxymethyl ester (calcein AM; 0.25 μM) was added for 30 min as the reporting molecule. After calcein AM was administered, it was cleaved by an esterase into a fluorescent product that is normally transported out of the cell by ABCB1. Positive controls were 100 μM verapamil and 50 μM digoxin. After the challenge, the TM cells were fixed at 4 °C in 3% paraformaldehyde for 15 min, mounted with Vectashield and 4',6-diamidino-2-phenylindole (DAPI) mounting medium, and analyzed by a masked observer using a Leica confocal microscope and software. RESULTS: Verapamil, an ABCB1 inhibitor, significantly (p<0.001) increased fluorescent calcein retention in the cytoplasm of the TM and RAW 264.7 cells compared to the PBS control. Digoxin, an ABCB1 activator, increased calcein efflux (p<0.001). Lactate reduced ABCB1 activity. HMW-HA significantly (p<0.001) reduced ABCB1 activity, whereas LMW-HA decreased ABCB1 activity, and the HA effects were blocked by naloxone (p<0.001), a TLR4 inhibitor. LPS alone did not change ABCB1 activity whereas dephosphorylated LPS significantly (p<0.001) enhanced ABCB1 activity in the TM cells. β-amyloid significantly reduced ABCB1 activity, and the β-amyloid effects were blocked by naloxone. CONCLUSIONS: TM cells are responsive to ABCB1 inhibitors and activators. ABCB1 functional activity is affected by TLR4 agonists suggesting that modulation of TLR4 is important in ABCB1 function. The innate immune inflammatory response in the TM may play a role in the ABCB1 detoxification of potentially harmful constituents in the aqueous humor.