Human Keratinocytes Constitutively Express Interleukin-18 and Secrete Biologically Active Interleukin-18 After Treatment with Pro-Inflammatory Mediators and Dinitrochlorobenzene  Shubhada M. Naik, Georgetta Cannon, Guido J. Burbach, Sareeta R. Singh, Robert A. Swerlick, John C. Ansel, S. Wright Caughman  Journal of Investigative Dermatology  Volume 113, Issue 5, Pages 766-772 (November 1999) DOI: 10.1046/j.1523-1747.1999.00750.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Human keratinocytes constitutively expressed IL-18 mRNA. Northern blot analyses of 10 μg total RNA isolated from either (A) human keratinocytes, or (B) HDMEC and HDF using a Klenow filled-in 32 P-labeled IL-18 cDNA to identify the IL-18 mRNA (upper panels) or β-actin cDNA to demonstrate even loading (lower panels).(A) Human keratinocytes that were either untreated (lanes 1 and 4) or treated with PMA (lane 2), LPS (lane 3), DMSO (lane 5) or DNCB (lane 6). (B) HDMEC and HDF that were either untreated (lanes 7 and 10) or treated with PMA (lanes 8 and 11) and LPS (lanes 9 and 12). Data are representative of three separate experiments. Journal of Investigative Dermatology 1999 113, 766-772DOI: (10.1046/j.1523-1747.1999.00750.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 IL-18 mRNA in human keratinocytes is associated with polyribosomes. Total RNA isolated from whole cell lysates of untreated human keratinocytes (5 × 106 cells) was analyzed by northern blot analysis to detect presence of IL-18 mRNA in either an aliquot of the lysate that was unfractionated (lane 1) or lysates that were fractionated on a 10–60% linear sucrose gradient (lanes 2–10). The RNA isolated from either the unfractionated (Unfx) or the various fractions containing the ribo- nucleoprotein complexes (RNP) and the polyribosomes associated complexes (polyribosomes) are indicated. Data are representative of three separate analyses. Journal of Investigative Dermatology 1999 113, 766-772DOI: (10.1046/j.1523-1747.1999.00750.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 IL-18 protein is constitutively expressed and secreted by human keratinocytes after treatment with PMA, LPS, or DNCB. Total protein from human keratinocyte lysates (A) or immunoprecipitated proteins from culture supernatants of THP.1, HK, and HDMEC using anti-IL-18 antibodies (B) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and specific proteins were detected by chemiluminescence as described in methods. IL-18, actin, and the molecular weight markers are indicated. These data are representative of three different experiments. (A) Western blot analysis of IL-18 protein in human keratinocyte lysates. Immunodetection of IL-18 protein (upper panel) and actin (lower panel) as an internal control is shown. Each lane was loaded with 50 μg of total proteins from human keratinocytes that were either untreated (lane 1) or treated with PMA (lane 2), DMSO (lane 3), or DNCB (lane 4) and subjected to western blot analysis. (B) IP/western blot analysis of IL-18 protein from THP.1, HK, and HDMEC supernatants. Two micrograms per milliliter of anti-IL-18 antibodies were used to immunoprecipitate proteins from 2.5 ml culture supernatants of THP.1 (lanes 1 and 2), HK (lanes 3–7), and HDMEC (lanes 8–12) that were either untreated (lanes 1, 3, and 8) or treated with DMSO (lanes 4 and 9), DNCB (lanes 5 and 10), LPS (lanes 6 and 11), or PMA (lanes 2, 7, and 12). Immunoprecipitated proteins were then subjected to western blot analysis for detection of IL-18 protein. Journal of Investigative Dermatology 1999 113, 766-772DOI: (10.1046/j.1523-1747.1999.00750.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Recombinant human IL-18 and a factor in treated human keratinocyte conditioned media induce IFN-γ expression by human PBMC. (A) rhIL-18 induces IFN-γ production by human PBMC in a concentration-dependent fashion. IFN-γ-specific ELISA was performed to detect production of IFN-γ by human PBMC that were either untreated (lane 1) or treated rhIL-18 for 24 h as follows: 1 ng per ml (lane 2); 10 ng per ml (lane 3); 100 ng per ml (lane 4), and 1000 ng per ml (lane 5). Values are expressed as pg per ml of IFN-γ produced by PBMC and represent the mean±SD of triplicate analyses. (B) Selected human keratinocyte conditioned media induce IFN-γ production by human PBMC. ELISA for IFN-γ expression was performed on normal keratinocyte media (KGM) or human keratinocyte culture supernatants (CS) prior to exposure to PBMC (left panel: –PBMC) or on CS after treatment of PBMC with selected human keratinocyte conditioned media for 24 h (right panel: +PBMC). ELISA results performed without PBMC (left panel) to determine whether human keratinocytes themselves secrete IFN-γ are displayed as follows: lane 1, KGM; lane 2, human keratinocyte CS after treatment with IL-18 (10 ng per ml); lane 3, human keratinocyte CS after treatment with PMA (10 ng per ml). ELISA results on PBMC supernatants (right panel) after treatment of PBMC with human keratinocyte CS are displayed as follows: lane 4, untreated human keratinocyte CS; lane 5, PMA (10 ng per ml for 16 h) treated human keratinocyte CS; lane 6, LPS (10 ng per ml) treated human keratinocyte CS; lane 7, DNCB (0.001%) treated human keratinocyte CS; lane 8, DMSO treated human keratinocyte CS. Values of detected IFN-γ are expressed as pg per ml as determined by internal rhIFN-γ standards and represent the mean±SD of triplicate analyses. Journal of Investigative Dermatology 1999 113, 766-772DOI: (10.1046/j.1523-1747.1999.00750.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Anti-hIL-18 but not anti-hIL-12 antibodies neutralize the IFN-γ-inducing factor secreted by human keratinocytes. IFN-γ-specific ELISA was performed to assess the ability of neutralizing antibodies to IL-18 and IL-12 to abrogate the ability of human keratinocyte CS and rhIL-18 to induce IFN-γ expression by PBMC. PBMC were exposed for 24 h to rhIL-18 and either untreated or PMA-treated (10 ng per ml) human keratinocyte CS as indicated below. rhIL-18 and human keratinocyte CS were incubated with neutralizing anti-IL-18 antibodies (8 μg per ml) or anti-IL-12 antibodies (1 μg per ml) for 30 min at 37°C prior to addition to PBMC. Variable treatments of PBMC with CS, rhIL-18, and antibodies are displayed in tabulated form above the ELISA data and are as follows: lane 1, untreated; lanes 2–4, rhIL-18 (100 ng per ml); lanes 3 and 7, anti-IL-18 antibody; lanes 4 and 8, anti-IL-12 antibodies; lane 5, untreated human keratinocyte CS; lanes 6–8, PMA-treated (10 ng per ml) human keratinocyte CS. Values of detected IFN-γ are expressed as pg per ml as determined by internal rhIFN-γ standards and represent the mean±SD of triplicate analyses. Journal of Investigative Dermatology 1999 113, 766-772DOI: (10.1046/j.1523-1747.1999.00750.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 In vivo expression of IL-18 in the human skin. Biopsied human skin sections from (A) normal human skin and (B) lesional psoriatic skin were subjected to immunohistochemistry for detection of in vivo expressed IL-18 and ICE proteins. (A) Immunodetection of normal human skin using primary antibodies to: (1) hIL-18; (2) ICE p20 subunit; and (3) normal goat serum control. (B) Immunodetection of skin sections from psoriatic lesional skin using primary antibodies to: (1) hIL-18; (2) ICE p20 subunit; and (3) normal goat serum control. Each panel is representative of skin biopsies from three normal and three psoriasis patients. Journal of Investigative Dermatology 1999 113, 766-772DOI: (10.1046/j.1523-1747.1999.00750.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions