July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Influence of serum from patients with Systemic Autoimmune Disease on IL-8 Expression in Human Corneal Epithelial Cells in Vitro.
Author Affiliations & Notes
  • Omer Iqbal
    Ophthalmology, Loyola University Chicago, Maywood, Illinois, United States
  • Avinash Kolli
    Ophthalmology, Loyola University Chicago, Maywood, Illinois, United States
  • Charles S Bouchard
    Ophthalmology, Loyola University Chicago, Maywood, Illinois, United States
  • Madeline Ripa
    Ophthalmology, Loyola University Chicago, Maywood, Illinois, United States
  • Adarsh Dharan
    Ophthalmology, Loyola University Chicago, Maywood, Illinois, United States
  • Simon Kaja
    Ophthalmology, Loyola University Chicago, Maywood, Illinois, United States
  • Edward Campbell
    Ophthalmology, Loyola University Chicago, Maywood, Illinois, United States
  • Footnotes
    Commercial Relationships   Omer Iqbal, None; Avinash Kolli, None; Charles Bouchard, None; Madeline Ripa, None; Adarsh Dharan, None; Simon Kaja, None; Edward Campbell, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4739. doi:
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      Omer Iqbal, Avinash Kolli, Charles S Bouchard, Madeline Ripa, Adarsh Dharan, Simon Kaja, Edward Campbell; Influence of serum from patients with Systemic Autoimmune Disease on IL-8 Expression in Human Corneal Epithelial Cells in Vitro.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4739.

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

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Abstract

Purpose : Systemic autoimmune disease such as Stevens Johnson Syndrome have ocular manifestations including dry eye, ocular surface inflammation and in severe cases corneal stem cell deficiency. Several proinflammatory cytokines such as TNF-α, IFN-¥, IL-6 and IL-8 have been shown to play a role in the pathogenesis of these conditions. Symptomatic management of dry eye disease include artificial lubricants, warm compresses, punctal occlusion, etc. It is hypothesized that autologous serum tears influence IL-8 expression in human corneal epithelial cells.

Methods : HCECs were subcultured in cell media. 150,000 cells per well were trypsinized and plated in a 12-well plate with a coverslip and cultured for 24 hours. The next day cells were exposed to TNF-α at concentrations of 0, 1, 10 and 100 ng/ml or AST for 16 hours, then incubated with IL-8 primary antibody in blocking solution for 1 hour. Subsequently, the cells were washed 3 times with 1XPBS and incubated with fluorescent secondary antibody in blocking solutions for 1 hour. Next, the cells were mounted onto DAPI mounting medium and allowed to cure overnight. Coverslips were imaged within 2-3 days after mounting. For quantitative PCR 300,000 cells per well were trypsinized, plated in a 12-well plate and cultured for 24 hours. Cells were then exposed to TNF-α at concentrations of 0, 1, 10, and 100 ng/ml or AST for 16 hours. Cells were lysed and Total RNA was extracted following the Nucleospin RNA plus kit. Subsequently, cDNA was created following the promega cDNA kit. qPCR was performed following the iTaq universal SYBR green protocol.

Results : TNF-α induced IL-8 expression in HCEC in a dose-dependent manner. This effect was not seen when IL-8 expression was associated with immunofluorescence microscopy. Autologous serum tears down regulated IL-8 expression in HCEC. This effect was independent of whether patient had autoimmune disease as a cause of their dry eye disease. There was no significant difference between HCEC treated with autologous serum tears derived from patients with systemic immune disease and autologous serum tears derived from normal.

Conclusions : Autologous serum tears can down regulate IL-8 expression in HCEC. Proinflammatory cytokine downregulation of inflammatory cytokines could be one mechanism of how autologous serum tears reduce signs and symptoms of patients with dry eyes.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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