Non-stimulated Tear Levels and Conjunctival Expression of IL-8 and IP-10 in Normal and Dry Eye Patients

Abstract

Purpose: : Levels of the pro-inflammatory tear chemokines IL-8 (CXCL8) and IP-10 (CXCL10) measured by antibody-based methods are often dismissed as artifact-prone, casting doubt on their usefulness as ocular surface disease biomarkers. In the current study, multiple methods were used to confirm tear levels and conjunctival surface expression of IL-8 and IP-10 and their associated receptors and biomarkers in dry eye patients and controls.

Methods: : A total of 25 patients, both normal and dry eye, participated in the study. Non-stimulated tear samples were collected by micropipette and stored undiluted at -80°C prior to either BioRad 27-Plex polystyrene bead-based cytokine assay on a Luminex 200 system or 1 D or 2 D SDS-PAGE, Odyssey Immunoblot, and Mass Spectrometry (MS) on an ABSciex 5600 Quadrupole-TOF mass spectrometer. Conjunctival cells were collected by impression cytology, RNA extracted by Qiagen RNEasy Plus Minikit, and real-time qPCR conducted on TaqMan 384-well low density array cards in 96A (96 gene) format.

Results: : Tear IL-8 and IP-10 excised from SDS-PAGE gels using matching Western blot localization, were both quantitatively identified in multiple samples by MS. In each case, a strongly ionizing peptide positively identified the chemokine. The average tear IP-10/IL-8 ratio averaged two times higher by 27-Plex Luminex assay than MS. Comparing tear chemokine levels with conjunctival gene expression further supported the tear Luminex assay results. Tear IL-8 levels correlated better with conjunctival surface expression of the IL-8Rβ receptor (CXCR2) than IL-8Rα (CXCR1), whereas conjunctival IL-8 expression correlated only with the IL-8Rα receptor (p < 0.001). Conjunctival IP-10 receptor (CXCR3) expression correlated more strongly with tear IP-10 than with conjunctival IP-10 expression. Conversely, conjunctival IFN-γ expression correlated strongly with IP-10 expression (p<0.001), whereas the associated tear levels were not correlated.

Conclusions: : The current study confirms the presence and relative levels of IL-8 and IP-10 in tears of both normal and dry eye patients by non-antibody-based methods and shows that valid tear levels are obtained by Luminex assay. Relationships between tear levels of IL-8 and IP-10, their conjunctival surface gene expression and conjunctival receptor gene expression further support the validity of the Luminex assay values and should help provide insights into their roles in dry eye.

Keywords: cornea: tears/tear film/dry eye • cytokines/chemokines • conjunctiva