Subtoxic levels hydrogen peroxide-induced production of interleukin-6 by retinal pigment epithelial cells

Abstract

Purpose: To study the effect of subtoxic levels of hydrogen peroxide (H(2)O(2)) on the expression and release of interleukin-6 (IL-6) by cultured retinal pigment epithelial (RPE) cells and to explore the relevant signal pathways.

Methods: Cultured human RPE cells were stimulated with various subtoxic concentrations of H(2)O(2) for different periods. Conditioned medium and cells were collected. IL-6 in the medium and IL-6 mRNA in the collected cells were measured using an IL-6 enzyme-linked immunosorbent assay kit and reverse transcriptase polymerase chain reaction, respectively. Nuclear factor-kappaB (NF-κB) in nuclear extracts and phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinases (JNK) in cells cultured with and without H(2)O(2) were measured by NF-κB and MAPK enzyme-linked immunosorbent assay kits. Inhibitors of p38 (SB203580), ERK (UO1026), JNK (SP600125), and NF-κB (BAY11-7082) were added to the cultures before the addition of H(2)O(2) to test their effects(.)

Results: Subtoxic levels of H(2)O(2) (100 µM and less) increased the IL-6 mRNA level and the release of IL-6 protein by the cultured human RPE cells in a dose- and time-dependent manner. This was accompanied by an increase of NF-κB in nuclear extracts and phosphorylated p38 MAPK, ERK, and JNK in cell lysates, particularly in the p38 and NF-κB. The NF-κB inhibitor decreased the H(2)O(2)-induced expression of IL-6. The p38 inhibitor, but not the ERK or JNK inhibitor, completely abolished H(2)O(2)-induced expression of IL-6 by RPE cells. The p38 inhibitor also abolished the increase of NF-κB in nuclear extracts in cells treated with H(2)O(2).

Conclusions: H(2)O(2) stimulated the production of IL-6, a key factor in the modulation of immune responses, inflammatory processes, and the occurrence of autoimmune diseases, which recently has been documented to be increased in age-related macular degeneration (AMD). This may be a molecular linkage for the oxidative stress and inflammatory/autoimmune reactions in AMD and may provide a novel target for the treatment of AMD.

Figure 1

The dose-dependent effects of subtoxic levels of hydrogen peroxide on release of interleukin-6 by retinal pigment epithelium cells. Retinal pigment epithelium (RPE) cells (ARPE-19) were plated into 24-well plates. After 24 h incubation, hydrogen peroxide (H₂O₂, at 0, 10, 30, 60, and 100 µM) was added to the culture and incubated for 24 h. Conditioned culture medium was collected and the amount of interleukin-6 (IL-6) protein in the conditioned medium was determined using the human IL-6 Quantikine enzyme-linked immunosorbent assay (ELISA) kit. IL-6 levels in conditioned culture medium were expressed as pg/ml (mean±standard deviation in triplicate tests). *0.01< p<0.05, **p<0.01, compared with the controls (cells cultured without H₂O₂).

Figure 2

The time-dependent effects of subtoxic levels of hydrogen peroxide on release of interleukin-6 by retinal pigment epithelium cells. Retinal pigment epithelium (RPE) cells (ARPE-19) were plated into 24-well plates. After 24 h incubation, 100 µM hydrogen peroxide (H₂O₂) was added to the culture and incubated for 2, 6, 12, and 24 h. Conditioned culture medium was collected and the amount of interleukin-6 (IL-6) protein in the conditioned medium was determined by using the human IL-6 Quantikine enzyme-linked immunosorbent assay (ELISA) kit. IL-6 levels in conditioned culture medium were expressed as pg/ml (mean±standard deviation in triplicate tests). *0.01< p<0.05, **p<0.01, compared with the controls (cells cultured without H₂O₂).

Figure 3

The effects of subtoxic levels of hydrogen peroxide on nuclear factor factor-kappaB in nuclear extracts and phosphorylated extracellular signal-regulated kinases, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. Retinal pigment epithelium (RPE) cells (ARPE-19) were plated into 24-well plates. After 24 h incubation, 100 µm hydrogen peroxide (H₂O₂) was added to the medium. Cells were collected 30 min later. The nuclear factor factor-kappaB (NF-κB) levels in nuclear extracts (A) and phosphorylated p38 mitogen-activated protein kinase (p38; B), c-Jun N-terminal kinase (JNK; C) and extracellular signal-regulated kinases (ERK; D) in cell lysates were measured using the relevant NF-κB enzyme-linked immunosorbent assay (ELISA) kit and phosphorylated MAPK ELISA kits (Biosource). The levels of NF-κB in nuclear extracts were expressed as pg/ml and phosphorylated p38, ERK and JNK in cell lysates were expressed as U/ml (mean±standard deviation in triplicate tests). *0.01< p<0.05, **p<0.01, compared with the controls (cells cultured without H₂O₂).

Figure 4

Effects of nuclear factor factor-kappaB and mitogen-activated protein kinase inhibitors on the hydrogen peroxide-induced production of interleukin-6 by retinal pigment epithelium cells. Retinal pigment epithelium (RPE) cells (ARPE-19) were plated into 24-well plates. After 24 h incubation, various mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) inhibitors were added to the medium separately, including BAY11–7082 (NF-κB inhibitor), UO1026 (extracellular signal-regulated kinases inhibitor), SP600125 (c-Jun N-terminal kinase inhibitor) and SB203580 (p38 MAPK inhibitor, all from Calbiochem) at a final concentration of 10 µM with the exception of BAY11–7082 (5 µM). After 30 min, 100 µM hydrogen peroxide (H₂O₂) was added to the medium. Cells cultured without H₂O₂ were used as negative controls. Cells cultured with H₂O₂ but without inhibitors were used as positive controls. After 24 h incubation, the culture medium was collected and the IL-6 levels were measured with the human IL-6 Quantikine ELISA kit and expressed as pg/ml (mean±standard deviation in triplicate tests). *0.01< p<0.05, **p<0.01, compared with the positive controls (cells cultured with H₂O₂ but without inhibitors).