Abstract
Purpose :
Due to the important role that myoepithelial cells (MEC) play in lacrimal gland contraction helping to expel lacrimal fluid, the purpose of the current study was to investigate if the proinflammatory cytokines, interleukin-1β (IL-1β), tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ), are implicated in the reported impaired MEC contraction in chronically inflamed lacrimal glands.
Methods :
MEC were isolated from lacrimal gland explants of α-Smooth Muscle Actin (SMA) - GFP mice (C57BL6) / SMACreErt2 strain strain. Cultured lacrimal gland MEC were treated with IL-1β alone (10 ng/ml) or a combination of IL-1β, TNFα and IFNγ (10 ng/ml) for a total of 7 days. Cells were maintained in complete RPMI-1640 medium (Roswell Park Memorial Institute) supplemented with 5% fetal bovine serum, and media was changed every other day with fresh addition of cytokines. At day 2, 4 and 7, GFP intensity, cell area, amounts of contractile proteins SMA and calponin, and MEC contraction were assessed. Analyzing GFP intensity is an indicator of SMA protein levels since GFP expression is under the control of the SMA promoter.
Results :
At day 0, control and treated cells showed no differences in GFP intensity and cell size. GFP intensity started to decrease in treated MEC at day 2 (20% ± 0.32; p=0.02), continuing at day 4 (25% ± 0.51; p=0.007) and day 7 (30% ± 0.48; p=0.0001). Mean cell area was also reduced at day 2 (34% ± 0.0001; p=0.0005), day 4 (51% ± 0.0003; p<0.0001) and day 7 (30% ± 0.001; p=0.0015). The contraction assay at day 2 showed a 70% ± 0.032 decrease of contraction in treated MEC compared with control (p<0.0001), 73% ± 0.008 (p<0.0001) at day 4 and a significant drop of 82% ± 0.006 (p=0.0015) at day 7. Also, at the last time point, levels of contractile proteins were measured by western blotting showing a decrease in the amount of SMA and calponin protein in treated MEC compared with the control group (30% ± 0.001; p=0.0016 and p=0.0206; respectively). Similar results were observed when tumor necrosis factor alpha (TNFα) or interferon gamma (IFNγ) were added along with IL-1β.
Conclusions :
Our results demonstrated that MEC contractile ability is impaired in the presence of pro-inflammatory cytokines IL-1β, TNFα, IFNγ making them potential therapeutic targets to restore MEC contractile ability and tear secretion in Sjogren syndrome dry eye disease.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.