Alarmins from conjunctival fibroblasts up-regulate matrix metalloproteinases in corneal fibroblasts.

Aim: To explore the effects of alarmins produced by necrotic human conjunctival fibroblasts on the release of matrix metalloproteinases (MMPs) by human corneal fibroblasts (HCFs).

Methods: A necrotic cell supernatant (NHCS) was prepared by subjecting human conjunctival fibroblasts to three cycles of freezing and thawing. The amounts of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in NHCS were determined by enzyme-linked immunosorbent assays. HCFs exposed to NHCS or other agents in culture were assayed for the release of MMPs as well as for intracellular signaling by immunoblot analysis. The abundance of MMP mRNAs in HCFs was examined by reverse transcription and real-time polymerase chain reaction analysis.

Results: NHCS increased the release of MMP-1 and MMP-3 by HCFs as well as the amounts of the corresponding mRNAs in the cells. NHCS also induced activation of mitogen-activated protein kinase (MAPK) signaling pathways mediated by extracellular signal-regulated kinase (ERK), p38, and c-Jun NH-terminal kinase (JNK) as well as elicited that of the nuclear factor (NF)-κB signaling pathway by promoting phosphorylation of the endogenous NF-κB inhibitor IκB-α. Inhibitors of MAPK and NF-κB signaling as well as IL-1 and TNF-α receptor antagonists attenuated the NHCS-induced release of MMP-1 and MMP-3 by HCFs. Furthermore, IL-1β and TNF-α were both detected in NHCS, and treatment of HCFs with these cytokines induced the release of MMP-1 and MMP-3 in a concentration-dependent manner.

Conclusion: Alarmins, including IL-1β and TNF-α, produced by necrotic human conjunctival fibroblasts triggered MMP release in HCFs through activation of MAPK and NF-κB signaling. IL-1β and TNF-α are therefore potential therapeutic targets for the amelioration of corneal stromal degradation in severe ocular burns.