Interaction of Interleukin–1 and Mechanical Stress in Mediating the Nuclear Factor Kappa B Stress Response in Trabecular Meshwork Cells

Abstract

Abstract: : Purpose: We have previously found that mechanical strain and shear stress enhance the ability of interleukin–1 (Il–1) to activate the NFΚB stress response in trabecular meshwork cells. The objective of this work was to determine the mechanism of this enhancing effect. Methods: Human trabecular meshwork cells were subjected to mechanical biaxial stretch (up to 10%) or shear stress (up to 0.5 dynes/cm²) before, during, or without the presence of IL–1α. Activation of NFΚB was determined by visualization of NFΚB in the nucleus by immunofluorescence microscopy and by Western immunoblotting of inhibitor kappa Bα (IΚB–α), which keeps NFΚB inactive in the cytoplasm by blocking its nuclear localization sequence. Secreted IL–1 was determined by enzyme–linked immunoabsorbant assay (ELISA). The role of calcium signaling in mechanically–enhanced NFΚB activation was examined using inhibitors of calcium mobilization. Results: Alone, neither mechanical strain nor subthreshold levels (0.03 ng/ml) of interleukin–1α induced NFΚB activation within the time period studied. However, the combination of mechanical strain or shear stress and IL–1α resulted in NFΚB activation within several hours of onset. Pre–stretching of trabecular meshwork cells for at least one hour prior to IL–1α addition, however, blocked the normal induction of NFΚB activation by IL–1α even at high levels (10 ng/ml). Culture media from mechanically–strained cells enhanced NFΚB activation by low levels of IL–1α. To investigate whether mechanical strain enhances the effect of IL–1α by inducing secretion of endogenous IL–1, we analyzed culture media of strained cells for IL–1 by ELISA. No IL–1α was detected but the sensitivity of the ELISA was inadequate to detect the predicted IL–1 levels (<10 pg/ml). BAPTA–AM, a chelator of free intracellular calcium, blocked NFΚB activation by the combination of shear stress and subthreshold IL–1, however, gadolinium, an inhibitor of stretch–sensitive cation channels, had no effect. Conclusions: Mechanical strain and shear stress can modulate the IL–1–induced NFΚB–mediated stress response that is characteristic of glaucomatous trabecular meshwork. This enhancing effect appears to be mediated by a product secreted by the strained cells and requires intracellular calcium signaling.

Keywords: trabecular meshwork • outflow: trabecular meshwork • stress response