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Fresia Carolina Lema, Carissa R Lumby, Rachel L Redfern; Effects of heat shock proteins in dry eye-associated inflammation in human corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1491.
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In dry eye disease, there is a vicious cycle of tear film hyperosmolar stress (HOS) and cytokines and matrix metalloproteinases (MMPs) production which can lead to damage on the ocular surface and potentially damage associated molecular patterns (DAMPs) release. Toll-like receptors (TLRs) are known to stimulate the production of cytokines and MMPs in response to pathogen associated molecular patterns (PAMPs) and potentially DAMPs either independently or synergistically with PAMPs. We investigated if HOS stimulates DAMPs release which in turn leads to the secretion of cytokines independently or in the presence of PAMPs in human corneal epithelial cells (HCEC).
Immortalized human SV40-B HCEC were cultured under hyperosmolar stress (400, 450 or 500mOsM) for 6, 12 and 24hr. The cell culture supernatants were collected and the level of DAMP, heat shock protein 70 (HSP70) was measured by ELISA. Further, HCEC cells were treated with increasing concentrations of HSP70 (0.001 to 1 µg/ml) with or without a PAMP (FSL-1, 1 µg/ml). After 6hr treatment, the cell culture supernatants were collected and Luminex assay quantified the secretion of 13 inflammatory cytokines. Results reflect the mean and standard deviation of three biological replicates.
Following 6hr exposure to HOS, only 500mOsM up-regulated HSP-70 secretion compared to untreated control (7,575 ± 1,038 pg/ml vs 985 ± 343 pg/ml; P ≤0.05). After 12hr, 450mOsM and 500mOsM induced a similar response (7,092±200 pg/ml and 6,625 ± 969 pg/ml, respectively vs 1,743 ± 856 pg/ml; P ≤0.05) and after 24hr of HOS treatment, there was no significant increase in HSP-70 compared to the untreated control. Data from Luminex assay showed that 6hr treatment with increasing concentrations of HSP-70 did not induce cytokine secretion, whereas FSL-1 alone significantly increased IL-6 and IL-8. Similarly, HSP70 with FSL-1 did not elicit significant cytokine secretion compared to the FSL-1 control.
Dry eye associated conditions (e.g. HOS) increased the release of HSP-70 in HCEC. However, at the concentrations and time-points tested, HSP-70 did not induce cytokine secretion either independently or in the presence of FSL-1 in HCEC. It remains to be determined if other DAMPs, which may be released in response to ocular surface damage are involved in stimulating inflammation in dry eye disease.
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