Involvement of reactive oxygen species in ionizing radiation-induced upregulation of cell surface Toll-like receptor 2 and 4 expression in human monocytic cells

Abstract

Toll-like receptors (TLRs) are pattern recognition receptors that recognize pathogen-associated molecular patterns and are indispensable for antibacterial and antiviral immunity. Our previous report showed that ionizing radiation increases the cell surface expressions of TLR2 and TLR4 and enhances their responses to agonists in human monocytic THP1 cells. The present study investigated how ionizing radiation increases the cell surface expressions of TLR2 and TLR4 in THP1 cells. The THP1 cells treated or not treated with pharmaceutical agents such as cycloheximide and N-acetyl-L-cysteine (NAC) were exposed to X-ray irradiation, following which the expressions of TLRs and mitogen-activated protein kinase were analyzed. X-ray irradiation increased the mRNA expressions of TLR2 and TLR4, and treatment with a protein synthesis inhibitor cycloheximide abolished the radiation-induced upregulation of their cell surface expressions. These results indicate that radiation increased those receptors through de novo protein synthesis. Furthermore, treatment with an antioxidant NAC suppressed not only the radiation-induced upregulation of cell surface expressions of TLR2 and TLR4, but also the radiation-induced activation of the c-Jun N-terminal kinase (JNK) pathway. Since it has been shown that the inhibitor for JNK can suppress the radiation-induced upregulation of TLR expression, the present results suggest that ionizing radiation increased the cell surface expressions of TLR2 and TLR4 through reactive oxygen species-mediated JNK activation.

Keywords: Toll-like receptor; c-Jun N-terminal kinase; ionizing radiation; reactive oxygen species.

Fig. 1.

Involvement of de novo protein synthesis in the radiation-induced upregulation of Toll-like receptor (TLR) 2 and TLR4 expressions. (A, B) THP1 cells treated with or without vehicle (H₂O) or cycloheximide (CHX) were exposed to 5 Gy. After 24 h of culture, the cells were harvested for analysis of cell surface expressions of TLR2 and TLR4. (A) Representative histograms of TLR2 and TLR4 expressions are shown. The dotted line and broken line indicate the isotype control and the result for non-irradiated cells, respectively. The black-filled histograms indicate the results for 5 Gy-irradiated cells. (B) Results are shown as the relative value of mean fluorescence intensity of irradiated cells compared with non-irradiated cells (5 Gy/0 Gy). Data are presented as the mean ± standard error (SE) of three independent experiments. (C) Non-irradiated or 5 Gy-irradiated THP1 cells were cultured for 12 h or 24 h, and total RNA was harvested for the analysis of the quantitative reverse transcription polymerase chain reaction. Data are presented as the mean ± SE of four independent experiments. * and ** indicate P < 0.05 and P < 0.01, respectively.

Fig. 2.

Effects of N-acetyl-L-cysteine (NAC) on the radiation-induced upregulation of Toll-like receptor (TLR) 2 and TLR4 expressions. (A) Upper panel: THP1 cells pretreated with vehicle (H₂O) or NAC for 1 h were harvested and washed with PBS(−). The cells were stained with 3′-(p-hydroxyphenyl) fluorescein (HPF), and then were exposed to 5 Gy in the presence of HPF. The cells were washed with PBS(−) immediately after irradiation and analyzed by a flow cytometer. Lower panel: NAC was added to the culture medium at 1 h before X-ray irradiation, following which the cells were exposed to 5 Gy. After 24 h of culture, the cells were harvested for the measurement of intracellular ROS levels. The dotted line indicates the result of the vehicle-treated non-irradiated control. Inset numbers indicate a relative value of mean fluorescence intensity compared with that of the vehicle-treated non-irradiated control. Representative results from two independent experiments are shown. (B) NAC was added to the culture medium at 1 h before X-ray irradiation, following which the cells were exposed to 5 Gy. After 24 h of culture, the cell surface expressions of TLR2 and TLR4 were analyzed. Results are shown as the relative value of mean fluorescence intensity compared with the vehicle-treated non-irradiated control. Data are presented as the mean ± standard error (SE) of three independent experiments. * indicates P < 0.01.

Fig. 3.

Effects of ceramide generation inhibitors on cell surface Toll-like receptor (TLR) 2 and TLR4 expressions. (A) THP1 cells were cultured in the presence of each ceramide generation inhibitor for 24 h and the cell surface TLR2 (left) and TLR4 (right) expressions were analyzed. As vehicle controls, cells treated with H₂O or 0.2% (v/v) dimethyl sulfoxide (DMSO) were prepared. Results are shown as a relative value of mean fluorescence intensity compared with that of the vehicle (H₂O) control. Data are presented as the mean ± standard error (SE) of three independent experiments. * and ** indicate P < 0.05 and P < 0.01, respectively. (B) Each ceramide generation inhibitor was added to the culture medium 1 h before X-ray irradiation, following which the cells were exposed to 5 Gy. After 24 h of culture, the cell surface expressions of TLR2 and TLR4 were analyzed. Results are shown as the relative value of mean fluorescence intensity compared with that of non-irradiated cells (5 Gy/0 Gy). Data are presented as the mean ± SE of three independent experiments.

Fig. 4.

Expression of mitogen-activated protein kinase (MAPK)–related proteins in X-ray–irradiated THP1 cells. (A) N-acetyl-L-cysteine (NAC) was added to the culture medium 1 h before X-ray irradiation, following which the cells were exposed to 5 Gy. After the indicated time-points, the cells were harvested for western blot analyses of phosphorylated c-Jun N-terminal kinase (p-JNK), phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated apoptosis signal-regulating kinase 1 (p-ASK1), ASK1, phosphorylated MAPK kinase 7 (p-MKK7) and phosphorylated c-Jun (p-c-Jun). As a loading control, the expressions of JNK, ERK or β-actin were analyzed. Representative data from at least two independent experiments are shown. (B) Two percent (v/v) dimethyl sulfoxide (DMSO) was added to the culture medium 1 h before X-ray irradiation, following which the cells were exposed to 5 Gy. After 1 h of culture, the cells were harvested for western blot analyses of phosphorylated JNK. As a loading control, the expression of JNK was analyzed. Representative data from two independent experiments are shown.