PURPOSE: The glycated human serum albumin (GHSA) has been demonstrated as effective chemokine inducer in human retinal pigment epithelial (hRPE) cells. Since little is known concerning endogenous chemokines induced by GHSA in corneal keratocytes, we investigated IL-8 and MCP-1 gene expression in human corneal keratocytes (HCK) as compared to human umbilical vein endothelial cells (HUVEC), a model for vascular endothelial cells, after stimulation by GHSA. METHODS: The HCK and HUVEC were incubated with GHSA, IL-1 beta and TNF-alpha. Secretion of IL-8 and MCP-1 was determined by enzyme-linked immunosorbent assay (ELISA). The total RNA was extracted from the corresponding cells and the mRNA levels were detected by Northern blot analysis. RESULTS: GHSA at 500 micrograms/ml concentration induced a significant increase in keratocyte IL-8 and MCP-1 protein secretion over the 24 h time course. The corresponding IL-8 mRNA levels reached a peak by 4 hr, while the MCP-1 mRNA increased steadily over this time period. The concentrations of GHSA for half-maximal stimulation of keratocyte IL-8 and MCP-1 secretion were 1,863 and 793 micrograms/ml, respectively. The levels of GHSA (500 micrograms/ml)-induced keratocyte IL-8 and MCP-1 expression were similar to that induced by IL-1 beta (0.02 ng/ml) and TNF-alpha (2.0 ng/ml). In contrast, the control HUVEC exposed to GHSA did not show sustained IL-8 and MCP-1 gene expression and protein secretion. CONCLUSIONS: This differential stimulation of keratocytes, not HUVEC, suggests that GHSA may be a plasma-borne inducer of chemokines acting on resident corneal cells at sites of inflammation where plasma leakage occurs.
PURPOSE: The glycated humanserum albumin (GHSA) has been demonstrated as effective chemokine inducer in humanretinal pigment epithelial (hRPE) cells. Since little is known concerning endogenous chemokines induced by GHSA in corneal keratocytes, we investigated IL-8 and MCP-1 gene expression in human corneal keratocytes (HCK) as compared to human umbilical vein endothelial cells (HUVEC), a model for vascular endothelial cells, after stimulation by GHSA. METHODS: The HCK and HUVEC were incubated with GHSA, IL-1 beta and TNF-alpha. Secretion of IL-8 and MCP-1 was determined by enzyme-linked immunosorbent assay (ELISA). The total RNA was extracted from the corresponding cells and the mRNA levels were detected by Northern blot analysis. RESULTS:GHSA at 500 micrograms/ml concentration induced a significant increase in keratocyte IL-8 and MCP-1 protein secretion over the 24 h time course. The corresponding IL-8 mRNA levels reached a peak by 4 hr, while the MCP-1 mRNA increased steadily over this time period. The concentrations of GHSA for half-maximal stimulation of keratocyte IL-8 and MCP-1 secretion were 1,863 and 793 micrograms/ml, respectively. The levels of GHSA (500 micrograms/ml)-induced keratocyte IL-8 and MCP-1 expression were similar to that induced by IL-1 beta (0.02 ng/ml) and TNF-alpha (2.0 ng/ml). In contrast, the control HUVEC exposed to GHSA did not show sustained IL-8 and MCP-1 gene expression and protein secretion. CONCLUSIONS: This differential stimulation of keratocytes, not HUVEC, suggests that GHSA may be a plasma-borne inducer of chemokines acting on resident corneal cells at sites of inflammation where plasma leakage occurs.
Authors: Matthew R Pennington; Amrita Saha; David F Painter; Christina Gavazzi; Ashrafali M Ismail; Xiaohong Zhou; James Chodosh; Jaya Rajaiya Journal: Microorganisms Date: 2019-09-13