Glucocorticoids Augment the Chemically Induced Production and Gene Expression of Interleukin-1α through NF-κB and AP-1 Activation in Murine Epidermal Cells  Yasuhiro Miyazaki, Hiroo Yokozeki, Sherif Awad, Ken Igawa, Kazuya Minatohara, Takahiro Satoh, Kiyoshi Nishioka  Journal of Investigative Dermatology  Volume 115, Issue 4, Pages 746-752 (October 2000) DOI: 10.1046/j.1523-1747.2000.00101.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Pattern of cytokine release in chemically stimulated keratinocytes. Pam 212 cells were incubated for 24 h with vehicle alone (Control) or with 0.1 mM TNBS, 0.1 mM DNBS, 0.01% MS, 10 μg per ml SDS, 1.0 μg per ml SEB in DMEM. The culture supernatants were collected and IL-1α, TNF-α, and IL-10 were measured by ELISA. Data represent the means ± SD of four independent experiments. A statistical analysis was performed using Student's t test, with *p < 0.05 and **p < 0.01 versus control (vehicle alone). Journal of Investigative Dermatology 2000 115, 746-752DOI: (10.1046/j.1523-1747.2000.00101.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Effect of hydrocortisone on the release of various cytokines by Pam 212 cells and epidermal cells stimulated with TNBS, DNBS, SDS, MS, and SEB. Pam 212 cells were incubated for 24 h with vehicle alone (Control) or with 0.1 mM TNBS, 0.1 mM DNBS, 0.01% MS, 10 μg per ml SDS, 1.0 μg per ml SEB in DMEM in the presence or absence of 10−10 M hydrocortisone. The culture supernatants were collected and IL-1α (A), TNF-α (B), and IL-10 (C) were measured by ELISA. The culture supernatants of TNBS-stimulated normal epidermal cells were measured IL-1α (D). The results represent the data of three independent experiments. Values are the mean ± SD of three determinations. A statistical analysis was done using Student's t test, with *p < 0.05 and **p < 0.01 versus TNBS treatment in the absence of hydrocortisone. Journal of Investigative Dermatology 2000 115, 746-752DOI: (10.1046/j.1523-1747.2000.00101.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Dose-response curve of IL-1α production from Pam 212 cells stimulated with haptens or irritants in the presence of various doses of hydrocortisone. Pam 212 cells were incubated for 24 h with 0.1 mM TNBS (A), 0.01% MS (B), 1 mg per ml SEB (C) in DMEM in the presence or absence of hydrocortisone (10−6−10−16 M). The culture supernatants were collected and IL-1α was measured by ELISA. The results represent the data of three independent experiments. Values are the mean ± SD of three determinations. A statistical analysis was done using Student's t test, with *p < 0.05 and **p < 0.01 versus the IL-1α release induced by 0.1 mM TNBS in the absence of hydrocortisone. Journal of Investigative Dermatology 2000 115, 746-752DOI: (10.1046/j.1523-1747.2000.00101.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The expression of IL-1α mRNA in Pam 212 cells stimulated with hapten in the presence or absence of hydrocortisone. Pam 212 cells were prepared as described in Materials and Methods. The cells were stimulated for 2 h with 0.1 mM TNBS and 0.1 mM DNBS in the presence or absence of hydrocortisone (A). Data are expressed as the relative densitometric values normalized to variations in β-actin from the same blot (B). The results represent the data of three independent experiments. Values are the mean ± SD of three determinations. A statistical analysis was done using Student's t test, with *p < 0.05 versus the IL-1α mRNA expression induced by 0.1 mM TNBS or 0.1 mM DNBS in the absence of hydrocortisone. Journal of Investigative Dermatology 2000 115, 746-752DOI: (10.1046/j.1523-1747.2000.00101.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Effect of hydrocortisone on nuclear translocation of the NF-κB p65 of Pam 212 cells stimulated with TNBS or DNBS. Pam 212 cells were incubated for 2 h with 0.1 mM TNBS (lanes 3, 4), 0.1 mM DNBS (lanes 5, 6) in DMEM in the presence (lanes 2, 4, 6) or absence (lanes 1, 3, 5) of 10−8 M hydrocortisone. Extract proteins (50 μg per lane) were separated by SDS-polyacrylamide gel electrophoresis, and then were electro-blotted to PVDF membranes. Immuno-staining of the membranes was performed with antisera specific for the p65 as the primary staining antibody and horseradish peroxidase-conjugated sheep antirabbit antiserum as the detection monoclonal antibody. Immunoblots were developed with the enhanced chemiluminescence Western blotting kit. These results represent the findings of three independent experiments. Journal of Investigative Dermatology 2000 115, 746-752DOI: (10.1046/j.1523-1747.2000.00101.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Activation of transcription factor NF-κB of Pam 212 cells stimulated with various chemicals. Pam 212 cells were treated with or without 0.1 mM TNBS, 0.1 mM DNBS, 0.0002% oxazolone, 10 μg per ml SDS, 0.01% MS, and 1.0 μg per ml SEB. Nuclear protein extracts were prepared and incubated with radiolabeled oligonucleotide probes. The resulting protein-DNA complexes were analyzed by gel EMSA (A). Pam 212 cells were treated with 50 μM PDTC for 1 h and 0.1 mM TNBS was then incubated for 2 h (B). More than 100-fold of oligonucleotides corresponding to the mutant version of the NF-κB motif were induced in the binding reaction (B). These results represent the findings of four independent experiments. Journal of Investigative Dermatology 2000 115, 746-752DOI: (10.1046/j.1523-1747.2000.00101.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Effect of hydrocortisone on the activity of transcription factor NF-κB of Pam 212 cells stimulated with TNBS. Pam 212 cells were treated with or without 0.1 mM TNBS in the presence or absence of hydrocortisone (10−6−10−10 M) (A). Nuclear protein extracts were prepared and incubated with radiolabeled oligonucleotide probes. The resulting protein-DNA complexes were analyzed by gel EMSA. (B) The super shift assays with monoclonal antibodies against p65. Pam 212 cells were stimulated with 0.01% MS (C), 1 mg per ml SEB (D) in the presence or absence of hydrocortisone (10−6−10−12 M). These results represent the findings of three independent experiments. Journal of Investigative Dermatology 2000 115, 746-752DOI: (10.1046/j.1523-1747.2000.00101.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Effect of PDTC on IL-1α release and the activity of transcription factor NF-κB of Pam 212 cells stimulated with TNBS and HC. Pam 212 cells were treated with 50 μM PDTC for 1 h, washed, and then incubated for 24 h with vehicle alone (Control) or with 0.1 mM TNBS in the presence or absence of 10−8 M hydrocortisone. Nuclear protein extracts were prepared and incubated with radiolabeled oligonucleotide probes. The resulting protein-DNA complexes were analyzed by gel EMSA. The culture supernatants were collected and IL-1α was measured by ELISA. The results represent the data of three independent experiments. Values are the mean ± SD of three determinations. A statistical analysis was done using Student's t test, with *p < 0.05 versus the IL-1α release from Pam 212 cells stimulated with TNBS plus hydrocortisone in the absence of PDTC. These results represent the findings of three independent experiments. Journal of Investigative Dermatology 2000 115, 746-752DOI: (10.1046/j.1523-1747.2000.00101.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Effect of hydrocortisone on the activity of transcription factor AP-1 of Pam 212 cells stimulated with TNBS. Pam 212 cells were treated with or without 0.1 mM TNBS in the presence or absence of hydrocortisone (10−6−10−12 M). Nuclear protein extracts were prepared and incubated with radiolabeled oligonucleotide probes. The resulting protein-DNA complexes were analyzed by gel EMSA. The figure shows that 10−8−10−10 M hydrocortisone enhanced the activation of AP-1 in TNBS-stimulated Pam 212 cells. These results represent the findings of three independent experiments. Journal of Investigative Dermatology 2000 115, 746-752DOI: (10.1046/j.1523-1747.2000.00101.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions