Abstract
Purpose:
Dry eye is characterized by hyperosmolar stress (HOS) on the ocular surface and secretion of inflammatory cytokines (IC) and metalloproteinases (MMPs) that cause disruption of the epithelium and potential release of heat shock proteins (HPSs) which may be involved in autoimmune conditions like Sjögren syndrome. Toll-like receptors (TLRs) are known to trigger the production of IC and MMPs in response to pathogen associated molecular patterns (PAMPs) while their ability to respond to HSPs is still unclear. We investigated the levels of secreted HSPs in human corneal epithelial cells (HCEC) under HOS and their ability to induce IC and MMPs production independently or synergistically with FSL-1, a PAMP recognized by TLR2.
Methods:
SV40 or primary HCEC were exposed to media, HOS (400- 500mOsM), or HSP70 (0.001 to 1 µg/ml) with or without FSL-1 (1 µg/ml) for up to 24hr. The fold change (FC) of secreted HSPs and concentration of 13 cytokines and 5 MMPs was determined in cell culture supernatants by luminex assays.
Results:
In SV40 HCEC, after 6hr HSP27 (FC=16.5±8.2), HSP60 (FC=35.3±5.3), HSP70 (FC=16.7±5.4) and HSP90 (FC+16.6±7.7) release was increased at 500mOsM; while at 450mOsM only HSP-60 (FC=12±6.4) showed significant increase. After 12hr, HSP-27 (FC=18.4±5.9), HSP70 (FC=36.3±16.2) and HSP90 (FC=3.7±0.3) were released at 500mOsM; and HSP27 (FC=17.6±5.9) and HSP70 (FC=20.1±12.1) were released at 450mOsM. After 24hr, the release of all HSPs drastically decreased and was non-significant when compared to the control. In Primary HCEC, after 6hr, levels of all HSPs were minimal and non-significant compared to the control. After 12hr, HSP27 was increased at 400(FC=5.2±2.5), 450(FC=4.7±1.1) and 500mOsM (11.9±3.9). Additionally, at 400mOsM and 450mOsM HSP60 (FC=4.2±1.1) and HSP90 (FC=4.1±1.3) were secreted, respectively. After 24hr, only HSP90 (FC=8.9±4.5) release at 500mOsM was significant. Treatment of SV40 HCEC with HSP70 for 24hr did not increase IC secretion when compared to the control; however in the presence of FSL-1, HSP70 did induce IL-6 and MMP-1 secretion.
Conclusions:
HSP27, HSP60, HSP70 and HSP90 increased up to 36.3 fold in SV40 HCEC and by 11.9 fold in primary HCEC in response to HOS. HSP70 may play a role in inflammation on the compromised ocular surface where there is an increase in microbial exposure and hyperosmolar stress.