Interleukin-17 and Interferon-γ Synergize in the Enhancement of Proinflammatory Cytokine Production by Human Keratinocytes  Marcel B.M. Teunissen, Jan D. Bos  Journal of Investigative Dermatology  Volume 111, Issue 4, Pages 645-649 (October 1998) DOI: 10.1046/j.1523-1747.1998.00347.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 IL-17 augments the IL-8 mRNA expression in keratinocytes. RNA was isolated from keratinocytes 8 h after stimulation with 0 (▪), 10 (♦), 50 (▴), or 250 (•) ng IL-17 per ml and was subjected to IL-8-specific RT-PCR, applying increasing numbers of PCR cycles. The radioactive amplification products were loaded onto an acryl amide gel and after electrophoresis the PCR products (left top) were scanned by a phosphorimager, generating densitometric values of each product, which were plotted against the PCR cycle numbers. Journal of Investigative Dermatology 1998 111, 645-649DOI: (10.1046/j.1523-1747.1998.00347.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 IL-17 concentration-dependently enhances the expression of IL-6 and IL-8 mRNA in keratinocytes. RNA was isolated from keratinocytes 8 h after stimulation with indicated concentrations of IL-17 and was subjected to semiquantitative cytokine-specific RT-PCR. The densitometric values of the IL-17-treated keratinocytes were related to the signal strength of the untreated keratinocytes, which was arbitrary set at 1. Journal of Investigative Dermatology 1998 111, 645-649DOI: (10.1046/j.1523-1747.1998.00347.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 IL-17 concentration-dependently enhances the secretion of IL-6 and IL-8 by keratinocytes. Supernatants from keratinocyte cultures were harvested 48 h after stimulation with indicated concentrations of IL-17 and were tested by ELISA for the presence of IL-1α, IL-6, IL-8, and IL-15. Data are mean results (± SD) of three experiments. Journal of Investigative Dermatology 1998 111, 645-649DOI: (10.1046/j.1523-1747.1998.00347.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 IL-17 and IFN-γ augment the IL-6 and IL-8 mRNA expression by keratinocytes. RNA was isolated from keratinocytes 8 h after stimulation with 50 ng IL-17 per ml, 100 U IFN-γ per ml, or both cytokines at the same time. The RNA was subjected to semiquantitative cytokine-specific RT-PCR. The densitometric values of the cytokine-treated keratinocytes were related to the signal strength of the untreated control keratinocytes (c), which was arbitrary set at 1. Journal of Investigative Dermatology 1998 111, 645-649DOI: (10.1046/j.1523-1747.1998.00347.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Synergism between IL-17 and IFN-γ in enhancement of IL-6 and IL-8 protein secretion by keratinocytes. Supernatants from keratinocyte cultures were collected 48 h after stimulation with 50 ng IL-17 per ml, 100 U IFN-γ per ml, or a cocktail of both cytokines, using supernatant of untreated keratinocytes (c) as a control. The concentration of IL-1α, IL-6, IL-8, and IL-15 in the supernatants was tested by ELISA. Data are mean results (± SD) of three experiments. Journal of Investigative Dermatology 1998 111, 645-649DOI: (10.1046/j.1523-1747.1998.00347.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 IL-17 weakly induces expression of ICAM-1 and HLA-DR on keratinocytes. After culture for 48 h in the absence (no IL-17) or presence of 50 ng IL-17 per ml, 100 U IFN-γ per ml, or both cytokines, keratinocytes were labeled with anti-ICAM-1 (top) or anti-HLA-DR (bottom). Presence of the cell surface markers on the keratinocytes was visualized with fluoroscein isothiocyanate-labeled anti-mouse antibodies and monitored by flow cytofluorometry. The isotype control for IL-17 treated keratinocytes is shown. The isotype controls for the IFN-γ and the IL-17 plus IFN-γ treated keratinocytes were identical to the isotype control in the figure (not shown). These data are representative of three independent experiments. Journal of Investigative Dermatology 1998 111, 645-649DOI: (10.1046/j.1523-1747.1998.00347.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 IL-17 mRNA is expressed by CD4+ and CD8+ psoriatic T cell clones. T cell clones from lesional psoriatic skin were cultured for 4 h with (+) or without (–) CD3/CD28 monoclonal antibody. RNA was isolated and subjected to RT-PCR to detect the presence of IL-17 mRNA. Detection of GAPDH was performed as a control for the quality of the RNA. After electrophoresis on a 1.4% agarose gel the PCR products were visualized by ethidium bromide staining. Clone P3/3 does express CD8, whereas the other four clones carry CD4. Lane M contains a DNA size marker, indicating the expected size of 407 bp of the IL-17 PCR product. Five representative clones out of 17 are shown. Journal of Investigative Dermatology 1998 111, 645-649DOI: (10.1046/j.1523-1747.1998.00347.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Presence of IL-17 mRNA in lesional psoriatic skin. Biopsies were taken from lesional (L) and nonlesional (NL) skin of three psoriasis patients (p#1, p#2, and p#3). RNA was isolated from cryostat sections and was subjected to RT-PCR to detect the presence of IL-17 mRNA. Detection of GAPDH was performed as a control for the quality of the RNA. After electrophoresis on a 1.4% agarose gel the PCR products were visualized by ethidium bromide staining. Lane M contains a DNA size marker and lane C contains the negative contol, in which the PCR reaction was performed in the absence of cDNA. Journal of Investigative Dermatology 1998 111, 645-649DOI: (10.1046/j.1523-1747.1998.00347.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions