Prostaglandin E2 (PGE2) induces IL-6 in human orbital fibroblasts through a CREB-dependent mechanism

Abstracts / Cytokine 48 (2009) 91–137 Secondary necrosis of apoptotic neutrophils induced by the human cathelicidin LL-37 is not proinflammatory to phagocytosing macrophages Hsin-Ni Li 1, Peter G. Barlow 1, Johan Bylund 2, Annie Mackellar 1, Åse Björstad 2, James Conlon 1, Pieter S. Hiemstra 3, Chris Haslett 1, Mohini Gray 1, A. John Simpson 1, Adriano G. Rossi 1, Donald J. Davidson 1, 1 MRC/ University of Edinburgh Centre for Inflammation Research, Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, 2 Department of Rheumatology, University of Gothenburg, Sweden, 3 Department of Pulmonology, Leiden University Medical Center, The Netherlands Cathelicidins are cationic host defence peptides (CHDP) with microbicidal, antiendotoxic, and multiple immunomodulatory properties. The importance of these peptides to innate host defence is indicated by the increased susceptibility to infection of individuals with morbus Kostmann and cathelicidin deficient mice, and the associations between cathelicidin levels and the pathogenesis of certain chronic diseases. Neutrophils (PMN) are the main reservoir of cathelicidins and play key roles in first line defence against infection. The appropriate regulation of PMN function, death, and clearance is critical to innate immunity and the efferocytosis of apoptotic PMN, in contrast to necrotic cells, is proposed to promote the resolution of inflammation. We demonstrate that the human cathelicidin LL-37 rapidly induced secondary necrosis of apoptotic human PMNs and identify the essential C-terminal region of LL-37 required this activity. LL-37-induced secondary necrosis did not affect PMN ingestion by human monocyte-derived macrophages and, in contrast to expectation, was not proinflammatory. The anti-inflammatory effects of apoptotic PMN on activated macrophages were retained and even potentiated in LL-37-induced secondarily necrotic PMN. In addition to the induction of secondary necrosis, LL-37 also promoted PMN granule contents release. Thus, we suggest that LL-37 induces swift secondary necrosis of PMN during inflammation without promoting macrophage inflammation, but may mediate host damage by releasing potentially harmful granules under chronic or dysregulated conditions.

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Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan, 4 Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan Endothelial progenitor cells (EPCs) are a circulating, bone marrow-derived cell population that appears to participate in vasculogenesis and tissue repair. However, the mechanisms by which EPCs home to remodeling tissues still remain unclear. Here, we show that CC chemokine receptor 5 (CCR5) is crucial for the homing of EPCs to skin wound in mice. We found that EPCs, characterized by CD34+/Flk-1+ cells, expressed CCR5 protein and existed in peripheral blood and skin tissue after wounding in wild-type (WT) mice. Although EPCs were mobilized from bone marrow into circulation in both WT and CCR5 / mice to a similar extent after wounding, the EPC homing to the wounds was significantly decreased in CCR5 / mice compared with that in WT mice. Consistent with this, CCR5 / mice exhibited attenuated neovascularization at the wound sites, which appeared to result in an impaired wound healing as evidenced by delayed wound closure and diminished granulation tissue formation. Transfer of BM cells from WT, but not from CCR5 / donor mice, restored wound healing along with augmented neovascularization in CCR5 / recipient mice. Moreover, injected EPCs migrated to the wounds and were incorporated into capillaries after systemic administration with wounding. We also confirmed that CC chemokine ligand CCL5, but not CCL3 and CCL4, induced migration of ex vivo expanded EPCs in a dose-dependent manner in vitro. Together, our data suggests that CCL5–CCR5 interaction are essential for skin wound healing through the homing of EPCs to wound. doi:10.1016/j.cyto.2009.07.486

doi:10.1016/j.cyto.2009.07.484

PP2-109 Using luminex (xMAP) technology to assay for inflammatory cytokines in cell culture supernatants from daudi and ovcar-3 cells treated with interferon

PP2-107 Absence of IFN-c accelerates thrombus resolution through enhanced MMP-9 and VEGF expression

Joseph Bekisz, Hana Schmeisser, David Stephany, Kathryn Zoon, Poster Presentation II Using luminex (xMAP) technology to assay for inflammatory cytokines in cell culture supernatants from daudi and ovcar-3 cells treated with interferon Joseph Bekisz, Hana Schmeisser, David Stephany, Kathryn Zoon, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

Toshikazu Kondo, Mizuho Nosaka, Yuko Ishida, Akihiko Kimura, Yumi Kuninaka, Masanori Inui, Naofumi Mukaida, Poster Presentation II Absence of IFN-gamma accelerates thrombus resolution through enhanced MMP9 and VEGF expression Toshikazu Kondo 1, Mizuho Nosaka 1, Yuko Ishida 1, Akihiko Kimura 1, Yumi Kuninaka 1, Masanori Inui 2, Naofumi Mukaida 1,2, 1 Department of Forensic Medicine, Wakayama Medical University, Wakayama, Japan, 2 Department of Immunology, Institute of Advanced Medicine We explored the pathophysiological roles of IFN-c in the resolution of venous thrombi. Upon the ligation of the inferior vena cava (IVC), venous thrombi developed progressively until 5 days, and remained similar sizes at 10 days. Concomitantly, intrathrombotic IFN-c contents were elevated progressively with an increase of post-ligation intervals. When IFN-c -deficient (IFN-c = ) mice were treated in the same manner, thrombus size was similar to that in wild-type (WT) mice until 5 days after the IVC ligation, but it was apparently smaller at 10 and 14 days, compared with WT mice. Intrathrombotic collagen-positive areas and intrathrombotic hydroxyproline contents were remarkably reduced later than 10 days after IVC ligation in IFNc = mice, compared with WT mice. However, there were no significant differences in intrathrombotic chemokine expression and leukocyte recruitment between both strains. Matrix metalloproteinase (MMP)-9 but not MMP-2 mRNA expression was higher at the late phase in IFN-c = mice, than WT mice. Concomitantly, MMP-9 enzyme activities were higher in IFN-c = mice, than WT mice. Moreover, intrathrombotic vascular areas were increased in IFN-c = mice, together with enhanced vascular endothelial growth factor (VEGF) gene expression, compared with WT mice. Furthermore, the administration of anti-IFN-c mAb accelerated the thrombus resolution in WT mice. Taken together, IFN-c can have a detrimental role in the thrombus resolution by suppressing MMP-9 and VEGF expression and can be a good molecular target for the treatment of deep vein thrombosis.

One goal of our research is to develop new interferon alpha constructs which will exhibit equal to or greater antiviral and antiproliferative activities but reduced side effects many of which may be related to inflammatory cytokines induced by the IFN treatment. Hybrid interferons composed of IFN-alpha 2c and IFN-alpha 21b were examined on Daudi (Burkitt’s Lymphoma) and OVCAR-3 (Ovarian carcinoma) cells for their antiviral and antiproliferative activities as well as the expression profiles of inflammatory cytokines and chemokines. Luminex (xMAP) technology was employed with this analysis which allows one to probe for a variety of analytes in a single sample and to quantitate the amount of that analyte in pg/ml. Three timepoints (24, 48 and 72 h) were examined at which cells were either left untreated or treated with IFN-a 2c (5 ng/ml). Cells culture supernatants were used for analysis. Preliminary results showed expected levels of IFN-alpha 2 given the amount of IFN-alpha used for treatment. Enhanced levels of IL-1 alpha were detected in both OVCAR-3 and Daudi samples, whereas IL-6 and IL-8 were upregulated in OVCAR-3 cells only. All three of these cytokines are associated with inflammation. Interestingly, the two macrophage inflammatory proteins (MIP-1a and MIP-1b) were upregulated in Daudi cells but not detected at all in IFN-treated OVCAR cells. TNF-a and TNF-b levels in both cell lines were essentially non-detectable. By far, the analyte expressed to the greatest extent in both cell lines was IP-10, a chemokine known to be induced by IFN-c and recently shown to be induced differentially by some IFN-a subtypes. Future work will include treating various cell lines with the IFN-a hybrids generated in our laboratory so that both the cell- specific and IFN-specific expression profiles can be determined and correlated with antiviral and antiproliferative activities and their ability to induce inflammatory cytokines and chemokines.

doi:10.1016/j.cyto.2009.07.485

doi:10.1016/j.cyto.2009.07.487

PP2-108 CCL5–CCR5 axis mediates skin wound healing by promoting endothelial progenitor cell accumulation

PP2-110 Prostaglandin E2 (PGE2) induces IL-6 in human orbital fibroblasts through a CREB-dependent mechanism

Yuko Ishida, Akihiko Kimura, Masanori Inui, Yumi Kuninaka, Kouji Matsushima, Naofumi Mukaida, Toshikazu Kondo, Poster Presentation II CCL5–CCR5 axis mediates skin wound healing by promoting endothelial progenitor cell accumulation Yuko Ishida 1, Akihiko Kimura 1, Masanori Inui 2, Yumi Kuninaka 1, Kouji Matsushima 3, Naofumi Mukaida 4, Toshikazu Kondo 1, 1 Department of Forensic Medicine, Wakayama Medical University, Wakayama, Japan, 2 Department of Molecular Immunology, Institute of Advanced Medicine, Wakayama Medical University, Wakayama, Japan, 3 Department of

Nupur Raychaudhuri, Terry J. Smith, Poster Presentation II Prostaglandin E2 (PGE2) induces IL-6 in human orbital fibroblasts through a CREBdependent mechanism Nupur Raychaudhuri, Terry J. Smith, Kellogg Eye Center, University of Michigan, Ann Arbor, MI, USA Background: IL-6 has been implicated in the pathogenesis of Graves’ disease and its inflammatory orbital manifestation, thyroid-associated ophthalmopathy (TAO).

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Abstracts / Cytokine 48 (2009) 91–137

Orbital fibroblasts produce extraordinarily high levels of PGE2, an important mediator of inflammation. Objective: To test the hypothesis that PGE2 induces IL-6 in orbital fibroblasts through cAMP response element binding protein (CREB) thus accounting for the anatomic-site specific expression of this cytokine. Design/methods: Fibroblast cultures were initiated from the orbits of patients with TAO or from normal skin. They were serum-starved and treated with nothing or PGE 2 (1 lM) for 16 h. IL-6 released into the medium was measured using an IL-6 ELISA and cell-layer-associated IL-6 was detected by Western blot. IL-6 mRNA was quantified using real-time PCR and gene promoter activity was assessed in fibroblasts transfected with 1168 to 3 bp fragment of the human IL-6 promoter fused to a luciferase reporter and measuring luminescence. Total and Ser-133-phosphorylated CREB (pCREB) were detected by Western blot. CREB was knocked down using siRNA. Results: PGE2 induces IL-6 synthesis and secretion in orbital fibroblasts far more robustly than in dermal cultures. This upregulation involved increased IL-6 mRNA through IL-6 gene promoter activity and could be mimicked with the PKA agonist Sp-cAMP. It could be blocked with the antagonists H89 and Rp-cAMP. This action is mediated through CREB. PGE2 provokes CREB phosphorylation. The pCREB/CREB ratio was disproportionately increased in orbit compared to dermal fibroblasts (p < 0.05) after PGE2 treatment. Fibroblasts transfected with CREB-targeting siRNA exhibited blunted IL-6 induction. Conclusion: CREB signaling is essential for the PGE2-induced IL-6 synthesis in orbital fibroblasts. These cells have been shown previously to express exaggerated levels of IL-6 and PGE2. We have now linked two important phenotypic attributes of orbital fibroblasts that may contribute to the pathogenesis of TAO. This finding suggests a potential target for therapeutic intervention.

S100A9 is a known pro-inflammatory protein abundantly expressed in the cytosol of neutrophils and monocytes. High extracellular S100A9 concentrations have been correlated with chronic inflammatory diseases such as rheumatoid arthritis and Crohn disease, as well as with phagocyte extravasation. In this study, we investigated the effect of S100A9 on human neutrophil degranulation. S100A9 was found to up-regulate the surface expression of CD35 and CD66b, proteins contained in secretory vesicles and specific/gelatinase granules, respectively. In addition, gelatinase and albumin, stored respectively in specific/gelatinase granules and secretory vesicles, were detected in the supernatants of neutrophils stimulated with S100A9. In contrast, stimulation with S100A9 had no effect on CD63 expression or myeloperoxidase secretion, two proteins contained in azurophilic granules. S100A9 induced to the phosphorylation of the mitogen-activated kinases ERK1/2, p38 and JNK. Inhibition of p38 and JNK, but not ERK1/2, with specific inhibitors (SB203580, JNKII and PD980549, respectively), blocked neutrophil degranulation induced by S100A9. Taken together, these results indicate that S100A9 induces the degranulation of secretory, specific and gelatinase granules, but not of azurophilic granules in a process involving p38 and JNK and further support its classification as a damage-associated molecular pattern.

doi:10.1016/j.cyto.2009.07.488

PP2-113 Myd88 gene knockout inhibits the development of lupus-like disease in NZB/W F1 mice

PP2-111 Involvement of Tsukushi in the pathogenesis of DSS-induced colitis in mice: Potential role of Tsukushi in inflammatory responses Kenji Watanabe, Shogo Shimasaki, Tsuyoshi Shuto, Mary Ann Suico, Tomoaki Koga, Takashi Sato, Kunimasa Ohta, Hirofumi Kai, Poster Presentation II Involvement of Tsukushi in the pathogenesis of DSS-induced colitis in mice: Potential role of Tsukushi in inflammatory responses Kenji Watanabe 1, Shogo Shimasaki 1, Tsuyoshi Shuto 1, Mary Ann Suico 1, Tomoaki Koga 1, Takashi Sato 1, Kunimasa Ohta 2, Hirofumi Kai 1, 1 Department of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Global COE ‘‘Cell Fate Regulation Research and Education Unit”, Kumamoto University, Kumamoto, Japan, 2 Department of Developmental Neurobiology, Graduate School of Medical Sciences, Global COE ‘‘Cell Fate Regulation Research and Education Unit”, Kumamoto University, Kumamoto, Japan Tsukushi is a member of the secreted small leucine-rich repeat proteoglycan (SLRP) family. Although the role of Tsukushi during developmental stages has been recognized, its biological role in the adult stage has not been elucidated in detail. Here, we show for the first time that a deficit in Tsukushi results in a greatly exaggerated response to dextran sodium sulfate (DSS)-induced colitis. Tsukushi / mice showed increase in clinical symptoms of inflammatory bowel disease (IBD) such as weight loss and the appearance of fecal blood at 7 days after 3% of DSS exposure. Consistently, gene expressions of TNF-a and KC, both of which are pro-inflammatory cytokines that contribute to IBD pathogenesis, were dramatically increased in the colon of DSS-treated Tsukushi / mice, whereas only slight increase of TNF-a and KC was observed in the colon of DSS-treated Tsukushi +/+ mice, suggesting a protective role of Tsukushi in the development of IBD pathogenesis. Notably, Tsukushi +/+ mice exhibited marked reduction of Tsukushi gene expression in the colon after DSS exposure, implying the possible involvement of Tsukushi for maintenance of cellular homeostasis during inflammation. Interestingly, despite a more severe phenotype in the colon of Tsukushi / mice compared with Tsukushi +/+ mice, cytokine responses of peritoneal macrophage to short-time exposure of several bacterial components (peptidoglycan, lipopolysaccharide, bacterial DNA, etc.) seemed identical among these mice. These results reveal the involvement of Tsukushi in the pathogenesis of DSS-induced colitis in mice and suggest a potential role of Tsukushi in immune and inflammatory responses. doi:10.1016/j.cyto.2009.07.489

PP2-112 Induction of neutrophil degranulation by S100A9 via a MAPK-dependent mechanism Jean-Christophe Simard, Denis Girard, Philippe A. Tessier, Poster Presentation II Induction of neutrophil degranulation by S100A9 via a MAPK-dependent mechanism Jean-Christophe Simard 1, Denis Girard 1, Philippe A. Tessier 2, 1 INRS-Institut ArmandFrappier, Université du Québec, Laval, Que., Canada, 2 Centre de Recherche en Infectiologie, Centre Hospitalier de l’Université Laval, and Faculty of Medicine, Laval University, Que., Canada

doi:10.1016/j.cyto.2009.07.490

Tomoharu Ohkawara, Minoru Fujimoto, Tetsuji Naka,Poster Presentation II Myd88 gene knockout inhibits the development of lupus-like disease in NZB/W F1 mice Tomoharu Ohkawara, Minoru Fujimoto, Tetsuji Naka The New Zealand Black(NZB)  New Zealand White (NZW) F1 hybrid female mice (NZB/WF1) develop a disease closely resembling human systemic lupus erythematosus (SLE), characterized by the production of antinuclear antibodies, severe glomerulonephritis and early mortality. Recently, innate immune responses have been reported to play an important role in the development of autoimmune disease. In this study we investigated the role of Myd88 protein (an important regulator of innate immune signaling pathways) in the pathogenesis of lupus-like disease in Myd88 knockout NZB/W F1 mice. Compared with WT mice, Myd88 KO mice featured significantly reduced serum concentrations of anti-dsDNA antibody, reduced levels of biochemical markers of active disease, reduced kidney pathology and reduced mortality. Our study demonstrates a critical role for Myd88 in the development of lupus-like disease in autoimmune mice. doi:10.1016/j.cyto.2009.07.491

PP2-114 The function of IL-17-producing cells in inflammatory disease Aoi Akitsu, Poster Presentation II The function of IL-17-producing cells in inflammatory disease Aoi Akitsu 1,2, Harumichi Ishigame 1, Shigeru Kakuta 1, Shinobu Saijo 1, Yoichiro Iwakura 1,2, 1 Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Japan, 2 Core Research for Evolutional Science and Technology (CREST), Japan IL-1 receptor antagonist-deficient (IL-1Ra KO) mice spontaneously develop autoimmune arthritis resembling rheumatoid arthritis in humans (Horai et al. J Exp Med, 2000). We previously demonstrated that IL-17 is involved in the development of the disease in this model (Nakae et al. PNAS, 2003). Furthermore, we showed that IL-1Ra deficiency in T cells plays an important role in the development of autoimmune arthritis (Horai et al. J Clin Invest, 2004). It is not known, however, that which IL17-producing cells are involved in the pathogenesis of arthritis in this model. Then, we analyzed IL-17-producing cells in these mice. We found that IL-17 production from both CD4+ T cells and gamma–delta T cells was significantly increased in draining lymph node from KO mice compared to wild-type mice, and that gamma–delta T cells, but not CD4+ cells, were the major IL-17 producers in the joints. When purified gamma–delta T cells from the spleen were treated with IL-1, IL-17 production was increased, suggesting that IL-17 production in gamma–delta T cells is directly induced by excess IL-1 signaling. Furthermore, IL-1Ra KO- nu/nu mice on the BALB/c background, who lacked thymus-derived T cells but still had gamma–delta T cells, developed arthritis. IL-17 producing-gamma–delta T cells in the draining lymph node of these mice were increased compared to those of control nu/nu mice, suggesting that extrathymus-derived T cells, such as IL-17 producing-gamma–delta T cells, have a pathogenic role in the development of arthritis. Taken together, these results suggest