Abstract
Purpose :
Vesicant injuries have been associated with chronic corneal pathology, including persistent inflammation and neovascularization. We and others have shown non-IgE-mediated activation of mast cells upon tissue injury leads to piecemeal release of inflammatory mediators. Here, we investigated the effect of alkylating agent exposure on mast cell activation and secretion of various inflammatory and chemotactic factors.
Methods :
Human mast cells (LAD2) were maintained in culture medium containing hSCF (100 ng/ml). To assess the direct effect of alkylating agents, mast cells were exposed directly to 10 µM of nitrogen mustard (NM) for 2 hours. To delineate whether NM-exposed human corneal epithelial cells (HCEC) activate mast cells, HCECs were pretreated with 10 µM of NM for 2 hours and the harvested supernatant were added to LAD2 cultures for additional 2h. To evaluate the continuous effect of NM exposure, mast cells were washed and incubated for another 6h in fresh media. Mast cell activation was evaluated by measuring level of β-hexosaminidase in the culture supernatant. Expression of inflammatory (TNFα, VEGF) and chemotactic factor (IL-8) were quantified in mustard-exposed mast cells. To evaluate the efficacy of mast cell inhibitor-cromolyn, NM-exposed mast cells were treated with 0.2% cromolyn.
Results :
Direct nitrogen mustard exposure of mast cells resulted in a 2-fold increase in levels of β-hexosaminidase, compared to media alone (p<0.001). Supernatants harvested from NM-exposed corneal epithelial cells also resulted in mast cell activation as evaluated by a significant upregulation of β-hexosaminidase levels (6-fold; p<0.001). Cromolyn treatment substantially suppressed the observed NM-induced mast cell activation (p=0.009, p=0.01, respectively). A significant increase in inflammatory factors TNFα (3-fold; p=0.029) and VEGF (p=0.005), and chemotactic factor IL-8 (>10-fold; p<0.001) was observed in mast cells following NM exposure. Moreover, inhibition of mast cells by cromolyn treatment significantly abrogated expression of both inflammatory and chemotactic factors (p<0.05).
Conclusions :
Our data demonstrate that direct exposure of nitrogen mustard and NM-exposed corneal epithelial cells activate mast cells to secrete inflammatory and chemotactic molecules, suggesting blockade of mast cells as a potential therapeutic strategy in attenuating alkylating agent-induced ocular inflammation and tissue damage.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.