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• W2051399616 abstract "Interleukin-18 is a potent inducer of interferon-γ by activated T cells, macrophages, and monocytes and is synthesized as an inactive precursor. Pro-interleukin-18 must be cleaved by interleukin-1-β-converting enzyme for secretion of the biologically active form. We report that among selected non-bone marrow derived skin cells, interleukin-18 mRNA is constitutively expressed by human keratinocytes and not by dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes. Interleukin-18 mRNA and intracellular protein levels are neither changed in human keratinocytes nor induced in human dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes by exposure to pro-inflammatory stimuli. Exposure of human keratinocytes to phorbol 12-myrisate 13-acetate, lipopolysaccharides or the contact sensitizer DNCB results in the secretion of immunoprecipitable interleukin-18 protein. Human keratinocyte-secreted interleukin-18 is biologically active, in that conditioned media from phorbol 12-myrisate 13-acetate, lipopolysaccharide and DNCB-treated human keratinocytes induce interferon-γ expression by peripheral blood mononuclear cells. This bioactivity is neutralized by anti-interleukin-18, but not anti-interleukin-12 antibodies. By immunohistochemistry, interleukin-18 protein is detected in basal keratinocytes of normal human skin, but its expression is markedly upregulated in suprabasal keratinocytes in psoriasis. These findings indicate that human keratinocytes are a source of biologically functional interleukin-18 and thus are capable of playing an initiating part in the local interferon-γ-dependent inflammatory processes through expression, activation, and secretion of interleukin-18. Interleukin-18 is a potent inducer of interferon-γ by activated T cells, macrophages, and monocytes and is synthesized as an inactive precursor. Pro-interleukin-18 must be cleaved by interleukin-1-β-converting enzyme for secretion of the biologically active form. We report that among selected non-bone marrow derived skin cells, interleukin-18 mRNA is constitutively expressed by human keratinocytes and not by dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes. Interleukin-18 mRNA and intracellular protein levels are neither changed in human keratinocytes nor induced in human dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes by exposure to pro-inflammatory stimuli. Exposure of human keratinocytes to phorbol 12-myrisate 13-acetate, lipopolysaccharides or the contact sensitizer DNCB results in the secretion of immunoprecipitable interleukin-18 protein. Human keratinocyte-secreted interleukin-18 is biologically active, in that conditioned media from phorbol 12-myrisate 13-acetate, lipopolysaccharide and DNCB-treated human keratinocytes induce interferon-γ expression by peripheral blood mononuclear cells. This bioactivity is neutralized by anti-interleukin-18, but not anti-interleukin-12 antibodies. By immunohistochemistry, interleukin-18 protein is detected in basal keratinocytes of normal human skin, but its expression is markedly upregulated in suprabasal keratinocytes in psoriasis. These findings indicate that human keratinocytes are a source of biologically functional interleukin-18 and thus are capable of playing an initiating part in the local interferon-γ-dependent inflammatory processes through expression, activation, and secretion of interleukin-18. human dermal microvascular endothelial cells human dermal fibroblasts human melanocytes interleukin-1β enzyme peripheral blood mononuclear cells The epidermal tissue of the skin is the first line of defense against exposure to physical, microbial, and chemical agents that cause local cellular injury (Kupper, 1990Kupper T.S. The role of epidermal cytokines in immunophysiology.in: Shevach E. Oppenheim J. The Role of Cells and Cytokines in Immunity and Inflammation. Oxford University Press, New York1990: 285-305Google Scholar;Kalish, 1995Kalish R.S. Poison ivy dermatitis: pathogenesis of allergic contact dermatitis to urushiol. Prog.Dermatol. 1995; 29: 1-7Google Scholar). During the evolution of an inflammatory response, leukocyte subsets are recruited and extravasate through the locally modified endothelium, a process that is orchestrated by local cytokine production (Springer, 1995Springer T.A. Traffic signals on the endothelium for lymphocyte recirculation and leukocyte emigration.Annu Rev Physiol. 1995; 57: 827-872Crossref PubMed Scopus (1392) Google Scholar;Ebnet et al., 1996Ebnet K. Kaldijian E.P. Anderson A.O. Shaw S. Orchestrated information transfer underlying leukocyte endothelial interactions.Annu Rev Immunol. 1996; 14: 155-177Crossref PubMed Scopus (181) Google Scholar). The activation of macrophages and T lymphocytes during the inflammatory responses elicits a concomitant release of cytokines by these cells, including interferon (IFN)-γ (Romagnani, 1994Romagnani S. Lymphokine production by human T cells in disease states.Annu Rev Immunol. 1994; 12: 227-257Crossref PubMed Google Scholar). The newly described cytokine interleukin (IL-18), also known as IFN-γ-inducing factor (IGIF), is a potent inducer of IFN-γ by activated T cells (Okamura et al., 1995Okamura H. Tsutsui H. Komatsu T. et al.Cloning of a new cytokine that induces IFN-γ production by T cells.Nature. 1995; 378: 88-91Crossref PubMed Scopus (2406) Google Scholar). Since its initial characterization, IL-18 has been found to have multiple other effects upon various cells involved in the inflammatory response (Okamura et al., 1995Okamura H. Tsutsui H. Komatsu T. et al.Cloning of a new cytokine that induces IFN-γ production by T cells.Nature. 1995; 378: 88-91Crossref PubMed Scopus (2406) Google Scholar;Tsutsui et al., 1996Tsutsui H. Nakanishi K. Matsui K. Higashino K. Okamura H. Miyazawa Y. Kaneda K. IFN-gamma-inducing factor (IGIF) up-regulates Fas ligand-mediated cytotoxic activity of murine natural killer cell clones.J Immunol. 1996; 157: 3967-3973PubMed Google Scholar;Ushio et al., 1996Ushio S. Namba M. Okura T. et al.Cloning of the cDNA for human IFN-γ-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein.J Immunol. 1996; 156: 4274-4279PubMed Google Scholar;Puren et al., 1998Puren A.J. Fantuzzi G. Gu Y. Su M.S.S. Dinarello C.A. Interleukin-18 (IFNγ-inducing factor) induces IL-8 and IL-1β via TNFα production from non-CD14+ human blood mononuclear cells.J Clin Invest. 1998; 101: 711-721Crossref PubMed Scopus (526) Google Scholar). IL-12 and IL-18 play important parts in the development of T helper type 1 (Th1) cells (Okamura et al., 1995Okamura H. Tsutsui H. Komatsu T. et al.Cloning of a new cytokine that induces IFN-γ production by T cells.Nature. 1995; 378: 88-91Crossref PubMed Scopus (2406) Google Scholar) and are synergistic in the induction of IFN-γ by T cells (Okamura et al., 1995Okamura H. Tsutsui H. Komatsu T. et al.Cloning of a new cytokine that induces IFN-γ production by T cells.Nature. 1995; 378: 88-91Crossref PubMed Scopus (2406) Google Scholar;Robinson et al., 1997Robinson D. Shibuya K. Mui A. et al.IGIF does not drive Th1 development but synergizes with IL-12 for interferon-γ production and activates IRAK and NF-κB.Immunity. 1997; 7: 571-581Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar). IL-18 exhibits an IL-1 signature-like sequences (Ushio et al., 1996Ushio S. Namba M. Okura T. et al.Cloning of the cDNA for human IFN-γ-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein.J Immunol. 1996; 156: 4274-4279PubMed Google Scholar) and contains trefoil structures similar to the IL-1 family of cytokines (Murzin et al., 1992Murzin A.G. Lesk A.M. Chothia C.J. beta-Trefoil fold, Patterns of structure and sequence in the Kunitz inhibitors interleukins-1 beta and 1 alpha and fibroblast growth.Factors J Mol Biol. 1992; 223: 531-543Crossref PubMed Scopus (306) Google Scholar;Bazan et al., 1996Bazan J.F. Timans J.C. Kaselein R.A. A newly defined interleukin-1?.Nature. 1996; 379: 591-???Crossref PubMed Scopus (262) Google Scholar). IL-18 and IL-1, however, share only 15–18% homology at the amino acid level and exhibit different biologic activities that are transmitted through specific receptors (Parnet et al., 1996Parnet P. Garka K.E. Bonnert T.P. Dower S.K. Sims J.E. IL-1Rrp is a novel receptor-like molecule similar to the type I interleukin-1 receptor and its homologues T1/ST2 and IL-1R AcP.J Biol Chem. 1996; 271: 3967-3970Crossref PubMed Scopus (218) Google Scholar;Matsumoto et al., 1997Matsumoto S. Tsuji-Takayama K. Aizawa Y. Koide K. Takeuchi M. Ohta T. Kurimoto M. Interleukin-18 activates NF-κB in murine T helper type 1 cells. Biochem.Biophys Res Commun. 1997; 234: 454-457Crossref PubMed Scopus (189) Google Scholar;Robinson et al., 1997Robinson D. Shibuya K. Mui A. et al.IGIF does not drive Th1 development but synergizes with IL-12 for interferon-γ production and activates IRAK and NF-κB.Immunity. 1997; 7: 571-581Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar;Torigoe et al., 1997Torigoe K. Ushio S. Okura T. et al.Purification and characterization of the human interleukin-18 receptor.J Biol Chem. 1997; 272: 25737-25742Crossref PubMed Scopus (434) Google Scholar;Dinarello et al., 1998Dinarello C.A. Novick D. Puren A.J. et al.Overview of interleukin-18: more than an IFN-γ-inducing factor.J Leuk Biol. 1998; 63: 658-664PubMed Google Scholar). The murine (Okamura et al., 1995Okamura H. Tsutsui H. Komatsu T. et al.Cloning of a new cytokine that induces IFN-γ production by T cells.Nature. 1995; 378: 88-91Crossref PubMed Scopus (2406) Google Scholar) and human IL-18 (Ushio et al., 1996Ushio S. Namba M. Okura T. et al.Cloning of the cDNA for human IFN-γ-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein.J Immunol. 1996; 156: 4274-4279PubMed Google Scholar) cDNA share a 65% homology. Human IL-18 (hIL-18) is synthesized as an inactive precursor form (pro-hIL-18) of 23 kDa that does not contain a known signal peptide sequence (Okamura et al., 1995Okamura H. Tsutsui H. Komatsu T. et al.Cloning of a new cytokine that induces IFN-γ production by T cells.Nature. 1995; 378: 88-91Crossref PubMed Scopus (2406) Google Scholar). Like the activation of IL-1β (Thornberry et al., 1992Thornberry N.A. Bull H.G. Calaycay J.R. et al.A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes.Nature. 1992; 356: 768-774Crossref PubMed Scopus (2207) Google Scholar), murine pro-IL-18 has been shown to be activated by IL-1β-converting enzyme (ICE or Caspase 1) by cleavage at the N-term-Asp35 to secrete a biologically functional form of 18.8 kDa (Ghayur et al., 1997Ghayur T. Banerjee S. Hugunin M. et al.Caspase-1 processes IFN-γ-inducing factor and regulates LPS-induced IFN-γ production.Nature. 1997; 386: 619-623Crossref PubMed Scopus (1043) Google Scholar;Gu et al., 1997Gu Y. Kuida K. Tsutsui H. et al.Activation of interferon-γ inducing factor mediated by interleukin-1β converting enzyme.Science. 1997; 275: 206-209Crossref PubMed Scopus (1021) Google Scholar). To date IL-18 has been reported to be produced by activated macrophages such as Kupffer cells, monocytes (Okamura et al., 1995Okamura H. Tsutsui H. Komatsu T. et al.Cloning of a new cytokine that induces IFN-γ production by T cells.Nature. 1995; 378: 88-91Crossref PubMed Scopus (2406) Google Scholar;Ushio et al., 1996Ushio S. Namba M. Okura T. et al.Cloning of the cDNA for human IFN-γ-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein.J Immunol. 1996; 156: 4274-4279PubMed Google Scholar), osteoblasts (Udagawa et al., 1997Udagawa N. Horwood N.J. Elliot J. et al.Interleukin-18 (interferon-gamma-inducing factor) is produced by osteoblasts and acts via granulocyte/macrophage colony-stimulating factor and not via interferon-gamma to inhibit osteoclast formation.J Exp Med. 1997; 185: 1005-1012Crossref PubMed Scopus (359) Google Scholar), and dendritic and Langerhans cells (Stoll et al., 1998Stoll S. Jonuleit H. Schmitt E. et al.Production of functional IL-18 by different subtypes of murine and human dendritic cells (DC): DC-derived IL-18 enhances IL-12-dependent TH1 development.Eur J Immunol. 1998; 28: 3231-3239Crossref PubMed Scopus (260) Google Scholar) in addition to T cells. Non-bone marrow derived cells of the skin have been shown to express a wide array of pro-inflammatory cytokines either constitutively or after a variety of stimuli (Kupper, 1990Kupper T.S. The role of epidermal cytokines in immunophysiology.in: Shevach E. Oppenheim J. The Role of Cells and Cytokines in Immunity and Inflammation. Oxford University Press, New York1990: 285-305Google Scholar;Luger and Schwarz, 1990Luger T.A. Schwarz T. Evidence of an epidermal cytokine network.J Invest Dermatol. 1990; 95: 100S-104SAbstract Full Text PDF PubMed Google Scholar). Consequently, we investigated the profile of IL-18 expression in cultured human cutaneous cells, including keratinocytes, dermal microvascular endothelial cells (HDMEC), dermal fibroblasts (HDF), and melanocytes (HM). Several significant reports related to the expression of ICE and bioactive IL-18 prompted us to investigate the effects of pro-inflammatory substances on the expression of IL-18 in human keratinocytes, HDMEC, HDF, and HM. These reports included secretion of IL-1β in response to urushiol (the immunogenic moiety of poison ivy) via activation by ICE (Boehm et al., 1996Boehm K.D. Yun J.K. Strohl K.P. Elmets C.A. In-situ changes in the relative abundance of human epidermal cytokine messenger RNAs following exposure to the poison ivy/oak contact allergen, urushiol. Exp.Dermatol. 1996; 5: 150-160Google Scholar;Zepter et al., 1997Zepter K. Haeffner A. Soohoo L.F. et al.Induction of biologically active IL-1β-converting enzyme and mature IL-1β in human keratinocytes by inflammatory and immunologic stimuli.J Immunol. 1997; 159: 6203-6208PubMed Google Scholar), modulation of ICE by activators and inhibitors of protein kinase C such as phorbol 12-myristate 13-acetate (PMA) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (Zepter et al., 1997Zepter K. Haeffner A. Soohoo L.F. et al.Induction of biologically active IL-1β-converting enzyme and mature IL-1β in human keratinocytes by inflammatory and immunologic stimuli.J Immunol. 1997; 159: 6203-6208PubMed Google Scholar), and production of bioactive IL-18 by murine keratinocytes (Stoll et al., 1997Stoll S. Mueller G. Kurimoto M. et al.Production of IL-18 (IFN-γ-inducing factor) messenger RNA and functional protein by murine keratinocytes.J Immunol. 1997; 159: 298-302PubMed Google Scholar). We report that among the non-bone marrow derived cells of the skin, only human keratinocytes constitutively express IL-18 mRNA and protein. On exposure to selected inflammatory mediators and the contact sensitizer DNCB IL-18 mRNA levels were not modulated further in human keratinocytes or induced de novo in HDMEC, HDF, or HM. In addition, upon treatment with pro-inflammatory mediators and DNCB, human keratinocytes secrete biologically active IL-18 that is capable of inducing IFN-γ production by human peripheral blood mononuclear cells (PBMC). Immunohistochemical studies reveal that IL-18 protein is constitutively expressed in basal keratinocytes in normal human skin and that its expression is markedly increased in suprabasal keratinocytes in psoriasis. These studies thus demonstrate that keratinocyte-derived IL-18 may play an important part in IFN-γ-dependent and T cell-mediated inflammation in the skin. Human PBMC, isolated as previously described (Kanof et al., 1991Kanof M.E. Smith P.D. Zola H. Preparation of human mononuclear cell populations and subpopulations.in: Coligan J.E. Kruisbeek A.M. Margulies D.H. Shevach E.M. Strober W. Current Protocols in Immunology. John Wiley, New York1991: 7.1.1-7.7.7Google Scholar) and THP.1, a human monocytic cell line (ATCC, Rockville, MD), were cultured in RPMI-1640 media containing 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 4500 mg glucose per l and 1500 mg sodium bicarbonate per l (Vita Cell, ATCC). PBMC were supplemented with 10% human serum (Irvine Scientific, Santa Ana, CA) and THP.1 with 10% fetal bovine serum (Atlanta Biologicals Inc., Norcross, GA). Human keratinocytes, HM, HDMEC, and HDF were isolated from neonatal foreskins at the Emory Skin Diseases Research Core Center as previously described (Boyce and Ham, 1985Boyce S.T. Ham R.G. Cultivation, frozen storage, and clonal growth of normal human epidermal keratinocytes in serum free.Media J Tissue Culture Methods. 1985; 9: 83-93Crossref Scopus (258) Google Scholar;Kubota et al., 1988Kubota Y. Kleinman H. Martin G.R. Lawley T.J. Role of laminin and basement membrane in the differentiation of human endothelial cells into capillary-like structures.J Cell Biol. 1988; 107: 1589-1598Crossref PubMed Scopus (981) Google Scholar). Human keratinocytes were cultured in keratinocyte growth media supplemented with bovine pituitary extract (Clonetics, Walkersville, MD). HDMEC were cultured in MCDB 131 media (Life Technologies, Inc., Grand Island, NY) supplemented with 30% normal human serum (Irvine Scientific), N6,2′-O-dibutyryl-cyclic adenosine monophosphate (5 × 10-5 M; Sigma), 100 U per ml penicillin, 0.25 mg per ml amphotericin B, and 10 μg per ml streptomycin (all from Life Technologies, Inc.). HM were cultured in Melanocyte Growth Medium Bulletkit (Clonetics). HDF were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 3 mM L-glutamine, 100 U per ml penicillin, 0.25 mg per ml amphotericin B, and 10 μg per ml streptomycin (all from Life Technologies, Inc.), and 10% fetal bovine serum (Hy-Clone Laboratories Inc., Logan, UT). Cell cultures were maintained at 37°C in humidified incubators with 5% CO2. Human keratinocytes were passaged at 60–70% confluence to avoid differentiation, using subculture reagents (Clonetics). Experiments with primary cells (human keratinocytes, HM, HDMEC, and HDF) were conducted with subconfluent cells at passage 3. This was performed for IL-18 mRNA levels after treatment of cells with a variety of pro-inflammatory mediators. These mediators were added directly to cells after a fresh media change and consisted of 10 ng per ml PMA, 50 ng per ml lipopolysaccharide (LPS), dimethylsulfoxide (DMSO), or 0.001% DNCB in DMSO (all from Sigma) for 16 h. Ten micrograms of total cellular RNA isolated from treated or untreated cells was analyzed by northern blot using either human IL-18 or β-actin cDNA as probes and were subjected to autoradiography on X-OMAT film (Eastman Kodak Co., Rochester, NY). The human IL-18 cDNA was generated by reverse transcription–polymerase chain reaction from PBMC total RNA using primers corresponding to the nucleotides 178–201 (5′-ATGGCTGCTGAACCAGTAGAAGAC-3′) and 735–759 (5′-CTAGTCTTCGTTTTGAACAGTGAAC-3′) of the published hIL-18 cDNA (Okamura et al., 1995Okamura H. Tsutsui H. Komatsu T. et al.Cloning of a new cytokine that induces IFN-γ production by T cells.Nature. 1995; 378: 88-91Crossref PubMed Scopus (2406) Google Scholar) using the Superscript pre-amplification system (Life Technologies, Inc.) according to the manufacturer’s protocol. IL-18 cDNA amplified by reverse transcription–polymerase chain reaction from total PBMC RNA was sequenced to confirm identity with the published hIL-18 cDNA sequence (Okamura et al., 1995Okamura H. Tsutsui H. Komatsu T. et al.Cloning of a new cytokine that induces IFN-γ production by T cells.Nature. 1995; 378: 88-91Crossref PubMed Scopus (2406) Google Scholar) and used to probe northern blots. Polysome fractions were obtained as described (Rogers and Munro, 1987Rogers J. Munro H. Translation of ferritin light and heavy subunit mRNAS is regulated by intracellular chelatable iron levels in rat hepatoma cells.Proc Natl Acad Sci USA. 1987; 84: 2277-22981Crossref PubMed Scopus (114) Google Scholar;Caruccio and Ross, 1994Caruccio N. Ross J. Purification of a human polyribosome-associated 3′ to 5′ exoribonuclease.J Biol Chem. 1994; 269: 31814-31821PubMed Google Scholar;Schalinske et al., 1998Schalinske K.L. Chen O.S. Eisenstein R.S. Iron differentially stimulates translation of mitochondrial aconitase and ferritin mRNAs in mammalian cells. Implications for iron regulatory proteins as regulators of mitochondrial citrate utilization.J Biol Chem. 1998; 273: 3740-3746Crossref PubMed Scopus (77) Google Scholar). In brief, whole cell lysates were prepared from untreated human keratinocyte cells (1 × 107 cells) by scraping the cells in 500 μl of ice-cold lysis buffer comprised of 150 mM KCl, 10 mM MgCl2, 150 mg cycloheximide per ml, 0.5 mg heparin per ml (all from Sigma), 10 mM Tris–Cl (pH 7.2) (Life Technologies, Inc.), 20 mM DTT, 0.5% NP-40 (all from United States Biochemical Corp., Cleveland, OH), and 100 U RNAsin per ml (Promega Corp., Madison, WI) using diethylpyrocarbonate (Sigma) treated and autoclaved water. Cells were left on ice for 10 min and then centrifuged for 10 min at 735 × g to separate nuclei. Supernatants were stored on ice. A linear 10–60% (by weight) sucrose gradient was poured in 12 ml Beckman poly-allomer tubes (14 × 89 mm) containing ultra-pure sucrose (Life Technologies, Inc.) in 20 mM HEPES (pH 7.2), 250 mM KCl, 10 mM MgCl2, 20 mM DTT, 150 μg cycloheximide per ml, 0.5 μg heparin per ml, and 10 U RNAsin per ml. A 50 μl aliquot of the post-nuclear human keratinocyte supernatant was saved as a control and the remainder was applied gently on to the linear sucrose gradient and centrifuged at 110,000 × g in a swinging bucket rotor for 4 h at 4°C. Total RNA was isolated from the unfractionated control sample and from 1.25 ml fractions that were collected from the gradient. The RNA samples were then analyzed for the presence of IL-18 mRNA by northern blot. Equal amounts of total protein (50 μg) from treated or untreated cells were analyzed by western blot analysis. Polyclonal N-terminal or C-terminal anti-human IL-18 antibodies (Research Diagnostics Inc., NJ) at a 1:100 dilution and donkey–anti-goat IgG horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at a 1:1500 dilution were used as primary and secondary antibodies respectively. Actin was used as an internal protein standard for loading control and was detected by mouse anti-actin (Chemicon International, Inc., Temecula, CA) primary antibodies at a 1:1000 dilution and goat anti-mouse Ig (BioRad) at 1:5000 dilution. Equal volumes of cell culture supernatants were used with anti-IL-18 antibodies at a concentration of 2 μg per ml for immunoprecipitation by standard methods, except using a modified immunoprecipitation buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM Tris (pH 7.4), 1 mM ethylenediamine tetraacetic acid, 2 mM ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 0.2 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5% NP-40. The immunoprecipitated proteins were subjected to a western blot analysis using anti-IL-18 antibodies or an irrelevant control antibody. Blots were developed using the chemiluminescent detection reagent ECL (Amersham Life Science Inc.) and exposed to X-OMAT film (Eastman Kodak Co.) to obtain a fluorograph. Paraffin-embedded tissue sections of 4μm thickness from biopsied human psoriatic lesions and normal skin were used after deparaffinization and rehydration according to the standard protocol (Jaffe and Raffeld, 1996Jaffe E.S. Raffeld M. Identification of cells in tissue sections.in: Coligan J.E. Kruisbeek A.M. Margulies D.H. Shevach E.M. Strober W. Current Protocols in Immunology. John Wiley, New York1996: 5.8.1-5.8.8Google Scholar). Sections were fixed in 0.4% formaldehyde and immersed in 0.1 M Na-citrate (Sigma) at 100°C for 1 min for antigen unmasking. The tissue sections were subjected to peroxidase immunostaining using VectaStain elite ABC kit (Vector Laboratories Inc., Burlingame, CA) according to the manufacturer’s recommendations. Normal rabbit serum from the VectaStain kit was used to avoid nonspecific binding of the secondary biotinylated antibody. Normal goat serum (Accurate Chemical & Scientific Corp., Westbury, CT) was used as a first step negative control and goat–anti-human IL-18 or goat–anti-human ICE (p20 subunit) antibodies (all from Research Diagnostics Inc.) were used as primary antibodies at a 1:20 dilution. The primary antibodies were detected using biotinylated rabbit–anti-goat secondary antibodies and a preformed avidin and biotinylated horseradish peroxidase complex from the VectaStain elite ABC kit. Sections were stained with the AEC substrate (Vector Laboratories Inc.), counterstained with Mayer’s hematoxylin, coverslipped and photographed with an Olympus C35AD-4 camera, mounted on an Olympus BH2 microscope. Secretion of bioactive IL-18 from human keratinocytes after varying treatments was determined by the ability of human keratinocyte supernatants to induce IFN-γ secretion from human PBMC. Human keratinocytes were left untreated or stimulated for 2 h with 10 ng PMA per ml, 10 ng phorbol 12,13-dibutyrate per ml, 50 ng LPS per ml, DMSO, or 0.001% DNCB in DMSO. To avoid a carry over of these substances to PBMC after the 2 h treatment period, human keratinocytes were washed four times with solution A (pH 7.4) containing 30 mM HEPES-NaOH buffer, 10 mM glucose, 3 mM KCl, 130 mM NaCl, 1 mM Na2HPO4&middot:7H2O, and 0.0033 mM phenol red (all from Sigma), and then replenished with normal keratinocyte growth media. Cells were then cultured for 16 h in 2.5 ml media per 100 mm tissue culture plate. Culture supernatants were harvested and 40% of the supernatant (1 ml) was used to treat PBMC. PBMC used in the bioassay were pre-stimulated for 16 h with concanavalin A (0.5 mg per ml, Sigma) and then centrifuged and resuspended in RPMI media with 20% human serum. 1 × 106 cells per ml were used for a 24 h treatment with 1 ml of either RPMI media or keratinocyte growth media alone as controls; RPMI media with the appropriate concentration of rhIL-18 (Research Diagnostics Inc.), rhIL-12 (R&D Systems Inc., Minneapolis, MN) or culture supernatants from treated or untreated human keratinocytes, to bring the final concentration of human serum to 10%. A human IFN-γ-specific ELISA was used to quantitate IFN-γ secreted by the PBMC in the media, according to the manufacturer’s recommendations (R&D Systems and Endogen Inc., Woburn, MA) To determine if IFN-γ secretion was specifically induced by IL-18 in the human keratinocyte supernatants, neutralizing antibodies to either rhIL-18 (Research Diagnostics Inc.) or to rhIL-12 (Transduction Labs, Lexington, KY) were added to human keratinocyte supernatants at the appropriate concentrations and incubated for 30 min at 37°C prior to human keratinocyte supernatant exposure to PBMC. rhIL-18 or rhIL-12 preincubated, with or without the neutralizing antibodies, was used as control treatments of PBMC. The autoradiographs of the northern blots and fluorographs of western blots were scanned on a La Cie flat bed scanner (La Cie Ltd, Beaverton, OR) utilizing Adobe Photoshop software (Adobe Systems, Inc., Mountain View, CA). Subsequently, all of the digitized images were imported into and labeled in Microsoft Power Point (Microsoft Corp., Redmond, WA) and printed on a high-resolution laser printer. Each autoradiograph and fluorograph figure represents a computer-generated image and each is typical of the original data in the context of relative band and background densities. The profile of constitutive and induced IL-18 mRNA expression by cultured HK, HDMEC, HDF, and HM was determined. Human keratinocytes displayed a robust constitutive IL-18 mRNA expression (Figure 1a). In contrast, constitutive IL-18 mRNA could not be detected in either HDMEC, HDF (Figure 1b) or HM (data not shown). Although previously implicated in the expression and secretion of bioactive IL-18 in various cell types, treatment of human keratinocytes, HDMEC, HDF, and HM with PMA and LPS resulted in no change in their respective constitutive IL-18 mRNA levels. In addition, human keratinocytes treated with DNCB had no effect on RNA levels. A variety of pro-inflammatory cytokines, growth factors, chemokines, and neuropeptides, including tumor necrosis factor-α, IL-1β, IL-4, IL-6, IL-8, IL-12, vascular endothelial growth factor, granulocyte macrophage colony-stimulating factor, basic fibroblast growth factor, transforming growth factor-α, substance P, substance K, and the contact sensitizer DNCB failed to induce IL-18 mRNA expression de novo in HDMEC (data not shown). Thus in the human skin, human keratinocytes but not HDMEC, HDF, or HM express constitutive levels of IL-18 mRNA. Moreover, IL-18 expression was not modulated further in human keratinocytes or induced de novo in HDMEC, HDF, or HM by pro-inflammatory mediators including PMA and LPS or the contact sensitizer DNCB. To determine if the constitutively expressed IL-18 mRNA in human keratinocytes is also being translated into IL-18 protein, we determined the polyribosome profile of IL-18 mRNA in cultured untreated human keratinocytes. As shown in (Figure 2), IL-18 mRNA was found to be associated with polyribosome fractions and not within the ribonucleoprotein fraction. Association of IL-18 mRNA with polyribosomes indicates that IL-18 mRNA is actively being translated into IL-18 protein in cultured, unstimulated human keratinocytes. Western blot analysis of total cellular proteins from human keratinocytes was performed using anti-IL-18 antibodies (Figure 3a). Lysates from untreated cells as well as cells treated with PMA, DMSO, or DNCB displayed constitutive IL-18 protein expression. These findings corroborate our data demonstrating constitutive polyri" @default.
• W2051399616 created "2016-06-24" @default.
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• W2051399616 date "1999-11-01" @default.
• W2051399616 modified "2023-09-25" @default.
• W2051399616 title "Human Keratinocytes Constitutively Express Interleukin-18 and Secrete Biologically Active Interleukin-18 After Treatment with Pro-Inflammatory Mediators and Dinitrochlorobenzene" @default.
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