Abstract
Purpose :
To investigate the regulatory effect of anti-inflammatory compounds used routinely for allergic eye disease (cyclosporine A and its analogues; glucocorticoids) using a human mast cell model in vitro.
Methods :
Human cord blood CD34+stem cells (105) were cultured in 200ul Stemspan™ serum-free medium containing SCF [100ng/ml] and IL-6 [50ng/ml], with IL-3 [1ng/ml] added during the first 14 days. On week 9-11, 10% fetal calf serum was added. At week 11, cells were characterised by flow cytometry (FacsCaliber, BD) for CD117 (c-kit) and FcεR1. For cross-linking, cells were incubated with IgE [4μg/ml; 16 hr] before anti-IgE Ab[25μg/ml] was added. Other stimuli included PMA, ionomycin, and toll-like receptor (TLR) ligands. Multiplex cytokine assays were performed to detect CXCL8, CCL2, CCL3, CCL4, CCL24, CCL26 by Luminex.
Results :
At week 10, HCBMC were immunophenotyped as 100% c-kit+ and 90% FcεR1+. In response to stimulation, the CBMC secreted CCL2, CCL3, CCL4, CXCL8, CCL24, CCL26 and CXCL9. The secretion profile was dependent on the mode of stimulation with significant increases in secretion of IL-8 in response to PMA±ionomycin, FcER1 cross-linking, and ligands for TLR-2, -3 and -4. There was a differential downregulation of CCL2, CCL3 and CXCL8 but not CCL4 by Fluticasone at (0.1-100 nM). In contrast, Loteprednol only downregulated secretion of CCL4 and CXCL8. Dexamethasone had no effect on CXCL8, but significantly downregulated CCL2, CCL3 and CCL4. Fluocinolone inhibited CCL2, CCL4 and CXCL8. Cyclosporin A analogues downregulated secretion of CCL2, CCL3 and CCL4 dose-dependently, with 10uM being the most potent. CsA was demonstrated to downregulate phosphorylated Erk1/2 and p38, in response to IgE-stimulation, but not in response to ionomycin.
Conclusions :
Using this in vitro human mast cell model, there was a differential immunoregulatory effect on chemokine secretion by a panel of anti-inflammatory compounds, with the effect of CsA dependent on Erk1/2 and p38 signalling.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.