The Immune-Modulating Cytokine and Endogenous Alarmin Interleukin-33 Is Upregulated in Skin Exposed to Inflammatory UVB Radiation
2011; Elsevier BV; Volume: 179; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2011.03.010
ISSN1525-2191
AutoresScott N. Byrne, Clare Beaugie, Clare O’Sullivan, S. Leighton, Gary M. Halliday,
Tópico(s)Eosinophilic Esophagitis
ResumoThe cellular and molecular mechanisms by which UV radiation modulates inflammation and immunity while simultaneously maintaining skin homeostasis is complex and not completely understood. Similar to the effects of UV, IL-33 has potent immune-modulating properties that are mediated by the downstream induction of cytokines and chemokines. We have discovered that exposure of mice in vivo or human skin samples ex vivo to inflammatory doses of UVB induced IL-33 expression within the epidermal and dermal skin layers. Using a combination of murine cell lines and primary human cells, we demonstrate that both UV and the oxidized lipid platelet activating factor induce IL-33 expression in keratinocytes and dermal fibroblasts. Highlighting the significance of these results, we found that administering IL-33 to mice in vivo suppressed the induction of Th1-mediated contact hypersensitivity responses. This may have consequences for skin cancer growth because UV-induced squamous cell carcinomas that evade immunological destruction were found to express significantly higher levels of IL-33. Finally, we demonstrate that dermal mast cells and skin-infiltrating neutrophils closely associate with UV-induced IL-33–expressing fibroblasts. Our results therefore identify and support a role for IL-33 as an important early danger signal produced in response to inflammation-inducing UV radiation. The cellular and molecular mechanisms by which UV radiation modulates inflammation and immunity while simultaneously maintaining skin homeostasis is complex and not completely understood. Similar to the effects of UV, IL-33 has potent immune-modulating properties that are mediated by the downstream induction of cytokines and chemokines. We have discovered that exposure of mice in vivo or human skin samples ex vivo to inflammatory doses of UVB induced IL-33 expression within the epidermal and dermal skin layers. Using a combination of murine cell lines and primary human cells, we demonstrate that both UV and the oxidized lipid platelet activating factor induce IL-33 expression in keratinocytes and dermal fibroblasts. Highlighting the significance of these results, we found that administering IL-33 to mice in vivo suppressed the induction of Th1-mediated contact hypersensitivity responses. This may have consequences for skin cancer growth because UV-induced squamous cell carcinomas that evade immunological destruction were found to express significantly higher levels of IL-33. Finally, we demonstrate that dermal mast cells and skin-infiltrating neutrophils closely associate with UV-induced IL-33–expressing fibroblasts. Our results therefore identify and support a role for IL-33 as an important early danger signal produced in response to inflammation-inducing UV radiation. The UV radiation contained in sunlight has wide-ranging immunosuppressive capabilities spanning both the induction and effector phases of an immune response. This property of UV is a key event in skin carcinogenesis1Kripke M.L. Fisher M.S. Immunologic parameters of ultraviolet carcinogenesis.J Natl Cancer Inst. 1976; 57: 211-215Crossref PubMed Scopus (275) Google Scholar; but it may also have beneficial effects, including protection from multiple sclerosis,2McMichael A.J. Hall A.J. Does immunosuppressive ultraviolet radiation explain the latitude gradient for multiple sclerosis?.Epidemiology. 1997; 8: 642-645Crossref PubMed Google Scholar enhancing allograft tolerance,3Ullrich S.E. 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The IL-1-like cytokine IL-33 is constitutively expressed in the nucleus of endothelial cells and epithelial cells in vivo: a novel ‘alarmin’?.PLoS ONE. 2008; 3: e3331Crossref PubMed Scopus (899) Google Scholar and is significantly upregulated in inflamed skin samples of atopic dermatitis patients.36Pushparaj P.N. Tay H.K. H'ng S.C. Pitman N. Xu D. McKenzie A. Liew F.Y. Melendez A.J. The cytokine interleukin-33 mediates anaphylactic shock.Proc Natl Acad Sci USA. 2009; 106: 9773-9778Crossref PubMed Scopus (248) Google Scholar It has been hypothesized that IL-33 acts as a novel “alarmin” that is released in the full-length active form after tissue damage.35Moussion C. Ortega N. Girard J.P. The IL-1-like cytokine IL-33 is constitutively expressed in the nucleus of endothelial cells and epithelial cells in vivo: a novel ‘alarmin’?.PLoS ONE. 2008; 3: e3331Crossref PubMed Scopus (899) Google Scholar, 37Cayrol C. Girard J.-P. The IL-1-like cytokine IL-33 is inactivated after maturation by caspase-1.Proc Natl Acad Sci USA. 2009; 106: 9021-9026Crossref PubMed Scopus (540) Google Scholar In this way, IL-33 acts as an endogenous danger signal that mediates the recruitment of innate immune cells to sites of infection or cellular damage. In support of this hypothesis, we show here that exposure to physiologically relevant doses of inflammatory UVB induces IL-33 in the epithelial layers of murine skin and upregulates IL-33 expression in human epidermis. UVB, but not UVA, radiation also induced de novo expression of IL-33 in dermal fibroblasts. UV-induced epithelial-derived skin tumors that evade immunological destruction also produced significant constitutive amounts of IL-33. IL-33–producing fibroblasts were surrounded by mast cells as well as newly recruited neutrophils, demonstrating the potential for UV-induced IL-33 to enhance innate immunity in the skin and to act as a unique molecular sensor of UV-induced damage. Female C57BL/6 mice (Animal Resource Centre, Perth, WA, Australia) or P2X7R−/− mice38Solle M. Labasi J. Perregaux D.G. Stam E. Petrushova N. Koller B.H. Griffiths R.J. Gabel C.A. Altered cytokine production in mice lacking P2X(7) receptors.J Biol Chem. 2001; 276: 125-132Crossref PubMed Scopus (778) Google Scholar (Centenary Institute; Sydney, Australia), aged 8 to 10 weeks at the start of experiments, were used with approval from the University of Sydney animal ethics committee. Shaved murine skin was exposed to UV and, at various times afterward, irradiated skin samples were collected and either snap frozen for real-time quantitative (q) RT-PCR analysis or were embedded in OCT and snap frozen for histological analysis. Murine embryonic fibroblasts (MEFs) originally derived from C57BL/6 embryos were cultured as previously described.39King N.J. Kesson A.M. Interferon-independent increases in class I major histocompatibility complex antigen expression follow flavivirus infection.J Gen Virol. 1988; 69: 2535-2543Crossref PubMed Scopus (60) Google Scholar The BALB/c-derived immortalized keratinocyte cell line PAM 212 was cultured as described elsewhere.40Kim T.Y. Kripke M.L. Ullrich S.E. Immunosuppression by factors released from UV-irradiated epidermal cells: selective effects on the generation of contact and delayed hypersensitivity after exposure to UVA or UVB radiation.J Invest Dermatol. 1990; 94: 26-32Abstract Full Text PDF PubMed Google Scholar Details of the two murine UV-induced skin tumor cell lines have been described by us previously.41Byrne S.N. Halliday G.M. Phagocytosis by dendritic cells rather than MHC II(high) macrophages is associated with skin tumour regression.Int J Cancer. 2003; 106: 736-744Crossref PubMed Scopus (19) Google Scholar Normal human skin was obtained from a 35-year-old, fair-skinned, female abdominal surgery patient. Numerous whole skin samples were cultured in complete RPMI-1640 before ex vivo UV exposure. Normal human fibroblasts and keratinocyte cultures were derived from a 35-year-old, fair-skinned, female breast reduction surgery patient and maintained in Stemline Keratinocyte Medium II with Stemline Keratinocyte Growth Supplement (Sigma, St. Louis, MO). Additional fibroblast cultures were derived from three separate human foreskin samples. Cells were used after the third passage. Details of the UV source, shaving, exposure of mice, and monitoring of the UV source have been described by us previously.18Byrne S.N. Spinks N. Halliday G.M. Ultraviolet-A irradiation of C57BL/6 mice suppresses systemic contact hypersensitivity or enhances secondary immunity depending on dose.J Invest Dermatol. 2002; 119: 858-864Crossref PubMed Scopus (61) Google Scholar Briefly, a 1000-W xenon arc solar simulator (Oriel, Stratford, CT) that expertly recreates the solar UV spectrum, was used to deliver an accurate dose of solar-simulated UV (ssUV; containing both UVA and UVB). In some experiments, spectra containing only UVA or only UVB were used where indicated. The same solar-simulated UV source was used for the in vitro studies. In these experiments, the various murine and human cell lines and primary cells were plated onto chamber slides (BD Falcon, Bedford, MA) at 80% confluence in 1 mL of their respective media. Twenty-four hours after incubation at 37°C in a 5% CO2 incubator, the media were removed and replaced with 250 μL PBS to ensure that the cells did not dry out, and then the cells or tissue were exposed to UV. The dose of UV used in each experiment is indicated in the respective figure legends. In some experiments, murine fibroblasts were incubated with either 1 μmol/L of carbamyl-PAF (cPAF) (Biomol, Enzo Life Sciences, Farmingdale, NY), 100 μg/mL cis-Urocanic Acid (cis-UCA; Sigma, St. Louis, MO), or PBS for either 1 hour or 4 hours. After UV exposure or treatment, cells were returned to the incubator in their respective media for an additional 24 hours before immunofluorescent analysis. In other experiments, 3.3 × 105 LK-2 regressor or 13–1 progressor tumor cells were plated into T25 flasks (representing 50% confluence). The cells were then grown as indicated in various concentrations of rIL-33–supplmented complete RPMI-1640 (10% fetal calf serum) for 3 days (corresponding to the time that the 13–1 control-treated cells reached 100% confluence). At this point, all of the cultures were trypsinized and total viable cells counted using a ViCell fully automated cell counter (Beckman Coulter, Brea, CA). Frozen skin tissue sections (7 μm) or Tris-buffered saline (TBS)-washed chamber slide–adherent cells were fixed for 10 minutes in methanol (4°C). Slides were treated with a biotin blocking kit (X0590; Dako, Carpinteria, CA) as per the manufacturer's instructions before blocking other proteins with 5% normal equine serum for 1 hour at 25°C. Slides were then incubated for 18 hours at 4°C with 10 μg/mL goat anti-mouse IL-33 (AF3626; R&D Systems, Minneapolis, MN), goat anti-human IL-33 (AF3625; R&D Systems), or purified goat IgG (AB-108-C; R&D Systems). After this, the TBS-washed slides were incubated for 1 hour at 37°C with a 1/200 biotin-conjugated donkey anti-goat IgG secondary antibody (705-066-147; Jackson Immunoresearch, West Grove, PA). The slides were then washed in TBS before a 30-minute incubation at 37°C in 1/1000 strepavidin Alexa Fluor 488 (S-11223; Molecular Probes, Carlsbad, CA). Finally, the slides were mounted and coverslipped in DAPI containing mounting medium (H-1500; Vectorshield, Vector Laboratories, Burlingame, CA) before being photographed immediately. In some experiments, the fluorescently labeled sections were photographed immediately before the removal of the coverslip and subsequent staining of mast cells using toluidine blue as we have described.42Byrne S.N. Limon-Flores A.Y. Ullrich S.E. Mast cell migration from the skin to the draining lymph nodes upon UV-irradiation represents a key step in the induction of immune suppression.J Immunol. 2008; 180: 4648-4655PubMed Google Scholar For the simultaneous immunofluorescence detection of IL-33 with CD45 or CD117, slides were first labeled for IL-33 as above, substituting the biotin-labeled detecting antibody for a chicken anti-goat Alexa Fluor 488 (Molecular Probes). These slides were then washed and biotin blocked as above before being incubated for 2 hours at 37°C with 10 μg/mL of BD Biosciences clone 30-F11 (for CD45), 10 μg/mL Biolegend clone 2B8 (for CD117), or 10 μg/mL of eBioscience rat IgG2b isotype control. Rat monoclonals were then visualized using 1/200 preabsorbed biotinylated goat anti-rat Ig (559286; BD Biosciences, San Diego, CA) for 1 hour at 37°C followed by 1/1000 strepavidin-AF594 (S-32356; Molecular Probes). The 1A8 rat anti-mouse Ly6G clone (BD Biosciences) was used at 10 μg/mL for 2 hours at 37°C to specifically identify neutrophils43Daley J.M. Thomay A.A. Connolly M.D. Reichner J.S. Albina J.E. Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice.J Leukoc Biol. 2008; 83: 64-70Crossref PubMed Scopus (813) Google Scholar in IL-33–stained skin sections. Specificity was confirmed using a rat IgG2a isotype control (clone R35-95; BD Biosciences). Labeled neutrophils were then visualized using 1/200 preabsorbed biotinylated goat anti-rat Ig (559286; BD Biosciences) for 1 hour at 37°C, followed by 1:4000 Strepavidin NL557 (R&D Systems). For the quantitative analysis of IL-33 intensity in fibroblasts, keratinocytes, and tumor cell lines, fluorescent images were acquired using an Olympus DP70 digital color microscope camera mounted on an Olympus BX40 fluorescence microscope (Olympus, VIC, Australia). Random fields of view were selected for acquisition of the DAPI (460 nm) followed by the IL-33 (520 nm) channel. Within each experimental repeat, exposure times were kept constant. For image analysis, IL-33 fluorescence was first converted to a gray-scale image for the measurement of mean gray intensity using Adobe Photoshop CS3 (Adobe). The number of DAPI-stained nuclei per image were then automatically counted using ImageJ software (http://rsbweb.nih.gov/ij/). An arbitrary unit of fluorescence intensity per cell was then calculated for each image and pooled for each experimental group. Intensities were then normalized to the indicated control groups for statistical analysis. An RNeasy Fibrous Tissue kit (Qiagen 74704, Valencia, CA) and RNeasy mini kit (Qiagen 74104) were used according to the manufacturers instructions to extract total RNA from skin and cultured cells, respectively. Details of the methods used have been described by us previously.44Stapelberg M.P. Williams R.B. Byrne S.N. Halliday G.M. The alternative complement pathway seems to be a UVA sensor that leads to systemic immunosuppression.J Invest Dermatol. 2009; 129: 2694-2701Crossref PubMed Scopus (30) Google Scholar The forward (5′-AACAGGCCTTCTTCGTCCTT-3′) and reverse (5′-GAACGCACAGGCGTTTTACT-3′) primers for IL-33 were designed using Primer345Rozen S. Skaletsky H.J. Primer3 on the WWW for general users and for biologist programmers.Methods Mol Biol. 2000; 132: 365-386Crossref PubMed Google Scholar and verified using a nucleotide BLAST (National Center for Biotechnology Information) search of total gene specificity. Four housekeeping genes [GAPDH, 18s, hypoxanthine phosphoribosyltransferase (HPRT), and ubiquitin] were tested. The gene 18s was not suitable, as it was found to be differentially regulated by UV in the skin. GAPDH (Fwd 5′-CCACCATGGAGAAGGCTGGGGCTC-3′; Rev 5′-AGTGATGGCATGGACTGTGGTCAT-3′) and HPRT (Fwd 5′-CTTTGCTGACCTGCTGGATT-3′; Rev 5′-TATGTCCCCCGTTGACTGAT-3′) were chosen for UV-irradiated skin and cell lines, respectively, as these were the most abundantly and reliably expressed housekeeping genes. Real-time qRT-PCR using 2× platinum SYBR Green qPCR SuperMix-UDB (Invitrogen, Carlsbad, CA) was performed on a Rotor-Gene RG-6000 PCR machine. Cycle threshold (CT) values for IL-33 were normalized to GAPDH or HPRT and analyzed with Rotor-Gene 6.0 software (Corbett Research, NSW, Australia), as we have previously described.42Byrne S.N. Limon-Flores A.Y. Ullrich S.E. Mast cell migration from the skin to the draining lymph nodes upon UV-irradiation represents a key step in the induction of immune suppression.J Immunol. 2008; 180: 4648-4655PubMed Google Scholar In addition to groups of UV-exposed mice, three other groups of mice were given three daily i.p. injections of recombinant IL-33 (0 μg, 0.1 μg, or 1 μg in 200 μL PBS; PeproTech, Rocky Hill, NJ). Mice were then immunized 24 hours later on the shaved, nonirradiated abdominal skin with 30 μL of the chemical hapten 2,4-dinitro-1-fluorobenzene (DNFB; 0.5% v/v in 4:1 acetone:olive oil; Sigma-Aldrich, St. Louis, MO). Five days later, the ears of the mice were challenged with 15 μL DNFB (0.25% v/v in the same diluent) per ear, applied evenly to the dorsal and ventral surfaces. Each experiment contained a nonimmunized, challenged-only irritant control group. Increases in ear thickness were calculated using measurements taken immediately before, 24, 48, and 72 hours subsequent to DNFB challenge. To determine whether UV-induced IL-33 is involved in UV-induced immune suppression, groups of mice were intradermally injected with 6 μg goat IgG or goat anti-mouse IL-33 (AF3626; R&D Systems) administered as 4 × 25 μL individual injections into the back skin. Immediately after this treatment, mice were exposed to 80 kJ/m2 UV. The anti–IL-33 and control injections were repeated 6 and 24 hours a
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