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Modulation Of Human Basophil Degranulation By Geranylgeranyl Compounds
194
Association Of IL-33 With Atherogenic Cytokines: A Link Between Allergic Disease and Atherosclerosis Dr. Misu Paul, MD1, Dr. Allison B. Reiss, MD2, Dr. Steven Carsons, MD3, Dr. Luz S. Fonacier, MD, FAAAAI4, Dr. Iryna Voloshyna, PhD2; 1Section of Allergy and Clinical Immunology, Department of Medicine, Winthrop University Hospital, mineola, NY, 2Winthrop Research Institute, Department of Medicine, Winthrop University Hospital, Mineola, NY, 3Division of Rheumatology, Allergy and Immunology, Department of Medicine, Winthrop University Hospital, Mineola, NY, 4Section of Allergy and Clinical Immunology, Department of Medicine, Winthrop University Hospital, Mineola, NY. RATIONALE: Previous work by our group indicated that interleukin (IL) —33 might play a dual role in allergic diseases functioning as an inducer of immune responses and promoting pro-atherogenic changes in lipid metabolism. We investigate here the effects of IL-33 on THP-1 macrophage activation and relationships between IL-33 and other pro-and antiinflammatory cytokines in plasma of healthy (HC) and atopic patients (AP) with allergic rhinitis and asthma. METHODS: THP-1 human-macrophages were incubated for 24h under the following conditions: (1) phosphate buffered saline control, (2) IL-33 at concentrations of 5, 10, 20 and 50ng/ml. Cell culture supernatants and plasma from atopic patients [AP] (n520) and healthy controls [HC] (n510) were analyzed for levels of IL-33, IFNg, TNFa, IL-1b, IL-17a, IL10, IL-2, IL-5 with Milliplex-kit. RESULTS: Exposure of THP-1 macrophages to IL-33 resulted in a concentration-dependent release of TNFa (393.7 pg/ml for 50ng/ml IL-33 vs. 208.7 pg/ml for control [P<0.05, n52]), IFNg (2.49 pg/ml vs. 1.7 pg/ ml) and IL-1b (188.93 pg/ml vs. 110.9 pg/ml [P<0.05, n52]). Similar pattern was detected in AP plasma compared to HC. Increasing concentrations of IL-33 in plasma strongly correlated with levels of IFNg (r50.85), TNFa (r50.9) and IL-17a (r50.94). CONCLUSIONS: IL—33 in-vitro and in AP plasma augments levels of the pro-inflammatory cytokines IFNg and TNFa. Strong correlation of IL-33 and IL-17 in AP is a possible link for IL-33 involvement in IL-17 mediated activation of the adaptive T-cell response and inflammatory-cytokine induction. The known atheroma-promoting nature of IFNg and TNFa could explain pro-atherogenic properties of AP plasma.
195
Modulation Of Human Basophil Degranulation By Geranylgeranyl Compounds Dr. Yuko Nakase1, Dr. Masao Yamaguchi1, Dr. Naoya Sugimoto1, Dr. Maho Suzukawa, MD2, Dr. Hiroyuki Tamiya1, Dr. Yasuhiro Kojima1, Dr. Hisanao Yoshihara1, Dr. Michio Kuramochi1, Dr. Hidenori Arai1, Dr. Hiroyuki Nagase1, Dr. Ken Ohta1,2; 1Teikyo University School of Medicine, Tokyo, Japan, 2National Hospital Organization Tokyo National Hospital, Tokyo, Japan. RATIONALE: Diverse pharmacological actions of geranylgeranyl (GG) compounds are only partially clarified so far. These compounds are reported to exert various beneficial effects in vivo on inflammatory diseases at skin or lung, and protection at gastric mucosa. GG compounds can also modulate cellular functions in vitro, including enhancement of mast cell degranulation. In this study, we assessed whether these compounds can affect the activation of human basophils. METHODS: Basophils were obtained from normal volunteers and preincubated with geranylgeranylacetone (GGA) at 1 ml/ml for 15 min at 37oC, washed, and stimulated with various secretagogues. RESULTS: Degranulation of basophils induced by anti-IgE antibody or PMA was significantly enhanced by GGA or geranylgeraniol. However, These compounds failed to affect basophil histamine release evoked by MCP-1 or ionophore A23187. Compounds with shorter structures such as geranylacetone of farnesol did not alter basophil degranulation. Simvastatin, an inhibitor of intracellular generation of GG compounds, suppressed basophil histamine release by anti-IgE antibody or PMA. CONCLUSIONS: These results suggest that human basophils are a target cell type of GG compounds. Although the precise role of their modulation of basophil functions is not clear, these compounds may orchestrate the in
vivo actions on various cells, both resident and inflammatory, resulting in beneficial outcomes in tissue damaging disorders.
196
Variation In mRNA Expresion Of GATA1 and PRG2 In Umbilical Cord Blood Following IL-5 Stimulation Ms. Vanessa N. Omana, MSc1, Mrs. Jenny Thiele, MSc2, Dr. Anne K. Ellis, MD, MSc, FAAAAI3; 1Queen’s University, ON, Canada, 2Queens University, Kingston, ON, Canada, 3Departments of Medicine and Biomedical & Molecular Science, Queen’s University, Kingston, ON, Canada. RATIONALE: GATA1 and PRG2 are key genes in eosinophil-lineage commitment. Our laboratory recently developed a multiplexed qPCR analysis of GATA1 and PRG2 mRNA expression in non-adherent mononuclear cells (NAMNCs) following IL-5 stimulation. We aimed to determine if differences exist in mRNA expression patterns of GATA1 and PRG2in the umbilical cord blood (UCB) of infants with attributable risk of atopy. METHODS: UCB was collected at birth and maternal atopic status confirmed by skin prick testing (SPT) (n5 28 atopic, n5 18 non-atopic). Mononuclear cells (MNCs) were isolated from the UCB samples, cryopreserved, then later thawed and depleted of adherent cells. NAMNCs were incubated with human recombinant interleukin 5 (rhIL-5; 1ng/mL) and collected at 0, 24, 48 and 72 hours post-stimulation. Multiplex qPCR was performed to measure GATA1 and PRG2mRNA expression at all time points. RESULTS: Stimulation of cord blood NAMNCs with IL-5 resulted in steady downregulation of GATA1 expression beginning at 24 hours. Conversely, there was an upregulation of the PRG2 gene expression, as previously demonstrated. However, there were no significant differences in the GATA1 and PRG2 gene expression patterns of UCB samples from infants born to atopic mothers vs. non-atopics. (GATA1 P 5 0.2285, 0.3166, and 0.1174 24h, 48h and 72h, respectively; PRG2P 5 0.1663, 0.6285, and 0.7612 at 24h, 48h and 72h). CONCLUSIONS: In this study, multiplexed qPCR analysis of GATA1 and PRG2 mRNA expression showed no significant differences between infants born to atopic versus non-atopic mothers, but enhanced understanding of eosinophilopoesis in UCB.
197
Mast Cells Drive Tissue Inflammation By Producing IL-33 That Orchestrates a Unique Basophil Phenotype Dr. Chia-Lin Hsu, PhD, Dr. Paul Bryce, PhD; Division of Allergy-Immunology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL. RATIONALE: Skin, an important environmental interface, protects against allergens that can trigger local or systemic anaphylactic reactions in allergic individuals. Skin-resident mast cells drive an early edema followed by a late antigen-driven tissue inflammation (ADTI) characterized by inflammatory cell recruitment. While histamine is important for the immediate edema after encountering the antigens, the mechanisms that control the inflammatory cell influx are still unclear. METHODS: A passive cutaneous model was used to study the ADTI. Mice were intradermally injected with PBS in one ear and DNP-specific IgE in the other followed by intravenous challenge with DNP-HSA. Ear swelling was measured and cell infiltration was determined by flow cytometry. Murine bone marrow-derived basophils (BMBs) were generated in vitro by culturing with IL-3 and activated with IgE/DNP or IL-33. RESULTS: In vitro, we showed that IgE/DNP primed BMBs to produce Th2-associated cytokines like IL-4 and CCL24, while IL-33 activated basophils to secrete neutrophil-recruiting chemokines, such as IL-6, CXCL1 and CXCL2. In vivo, we showed that mast cell-derived IL-33 was sufficient and necessary to induce the late phase ear swelling. ST2 and IL-6-expressing basophils were also required for inflammatory cell recruitment. Furthermore, it was H4R responsible for basophil recruitment. CONCLUSIONS: Our finding indicated that mast cells acted as local sensors in responding to foreign antigens. Histamine was produced immediately and basophils were recruited through H4R. Migrated basophils were then activated by mast cell-derived IL-33 through ST2 and produced IL-6 to promote antigen-driven tissue inflammation by recruiting CD45+Gr-1high neutrophils.
SATURDAY
Abstracts AB55
J ALLERGY CLIN IMMUNOL VOLUME 133, NUMBER 2
Association Of IL-33 With Atherogenic Cytokines: A Link Between Allergic Disease and Atherosclerosis Dr. Misu Paul, MD1, Dr. Allison B. Reiss, MD2, Dr. Steven Carsons, MD3, Dr. Luz S. Fonacier, MD, FAAAAI4, Dr. Iryna Voloshyna, PhD2; 1Section of Allergy and Clinical Immunology, Department of Medicine, Winthrop University Hospital, mineola, NY, 2Winthrop Research Institute, Department of Medicine, Winthrop University Hospital, Mineola, NY, 3Division of Rheumatology, Allergy and Immunology, Department of Medicine, Winthrop University Hospital, Mineola, NY, 4Section of Allergy and Clinical Immunology, Department of Medicine, Winthrop University Hospital, Mineola, NY. RATIONALE: Previous work by our group indicated that interleukin (IL) —33 might play a dual role in allergic diseases functioning as an inducer of immune responses and promoting pro-atherogenic changes in lipid metabolism. We investigate here the effects of IL-33 on THP-1 macrophage activation and relationships between IL-33 and other pro-and antiinflammatory cytokines in plasma of healthy (HC) and atopic patients (AP) with allergic rhinitis and asthma. METHODS: THP-1 human-macrophages were incubated for 24h under the following conditions: (1) phosphate buffered saline control, (2) IL-33 at concentrations of 5, 10, 20 and 50ng/ml. Cell culture supernatants and plasma from atopic patients [AP] (n520) and healthy controls [HC] (n510) were analyzed for levels of IL-33, IFNg, TNFa, IL-1b, IL-17a, IL10, IL-2, IL-5 with Milliplex-kit. RESULTS: Exposure of THP-1 macrophages to IL-33 resulted in a concentration-dependent release of TNFa (393.7 pg/ml for 50ng/ml IL-33 vs. 208.7 pg/ml for control [P<0.05, n52]), IFNg (2.49 pg/ml vs. 1.7 pg/ ml) and IL-1b (188.93 pg/ml vs. 110.9 pg/ml [P<0.05, n52]). Similar pattern was detected in AP plasma compared to HC. Increasing concentrations of IL-33 in plasma strongly correlated with levels of IFNg (r50.85), TNFa (r50.9) and IL-17a (r50.94). CONCLUSIONS: IL—33 in-vitro and in AP plasma augments levels of the pro-inflammatory cytokines IFNg and TNFa. Strong correlation of IL-33 and IL-17 in AP is a possible link for IL-33 involvement in IL-17 mediated activation of the adaptive T-cell response and inflammatory-cytokine induction. The known atheroma-promoting nature of IFNg and TNFa could explain pro-atherogenic properties of AP plasma.
195
Modulation Of Human Basophil Degranulation By Geranylgeranyl Compounds Dr. Yuko Nakase1, Dr. Masao Yamaguchi1, Dr. Naoya Sugimoto1, Dr. Maho Suzukawa, MD2, Dr. Hiroyuki Tamiya1, Dr. Yasuhiro Kojima1, Dr. Hisanao Yoshihara1, Dr. Michio Kuramochi1, Dr. Hidenori Arai1, Dr. Hiroyuki Nagase1, Dr. Ken Ohta1,2; 1Teikyo University School of Medicine, Tokyo, Japan, 2National Hospital Organization Tokyo National Hospital, Tokyo, Japan. RATIONALE: Diverse pharmacological actions of geranylgeranyl (GG) compounds are only partially clarified so far. These compounds are reported to exert various beneficial effects in vivo on inflammatory diseases at skin or lung, and protection at gastric mucosa. GG compounds can also modulate cellular functions in vitro, including enhancement of mast cell degranulation. In this study, we assessed whether these compounds can affect the activation of human basophils. METHODS: Basophils were obtained from normal volunteers and preincubated with geranylgeranylacetone (GGA) at 1 ml/ml for 15 min at 37oC, washed, and stimulated with various secretagogues. RESULTS: Degranulation of basophils induced by anti-IgE antibody or PMA was significantly enhanced by GGA or geranylgeraniol. However, These compounds failed to affect basophil histamine release evoked by MCP-1 or ionophore A23187. Compounds with shorter structures such as geranylacetone of farnesol did not alter basophil degranulation. Simvastatin, an inhibitor of intracellular generation of GG compounds, suppressed basophil histamine release by anti-IgE antibody or PMA. CONCLUSIONS: These results suggest that human basophils are a target cell type of GG compounds. Although the precise role of their modulation of basophil functions is not clear, these compounds may orchestrate the in
vivo actions on various cells, both resident and inflammatory, resulting in beneficial outcomes in tissue damaging disorders.
196
Variation In mRNA Expresion Of GATA1 and PRG2 In Umbilical Cord Blood Following IL-5 Stimulation Ms. Vanessa N. Omana, MSc1, Mrs. Jenny Thiele, MSc2, Dr. Anne K. Ellis, MD, MSc, FAAAAI3; 1Queen’s University, ON, Canada, 2Queens University, Kingston, ON, Canada, 3Departments of Medicine and Biomedical & Molecular Science, Queen’s University, Kingston, ON, Canada. RATIONALE: GATA1 and PRG2 are key genes in eosinophil-lineage commitment. Our laboratory recently developed a multiplexed qPCR analysis of GATA1 and PRG2 mRNA expression in non-adherent mononuclear cells (NAMNCs) following IL-5 stimulation. We aimed to determine if differences exist in mRNA expression patterns of GATA1 and PRG2in the umbilical cord blood (UCB) of infants with attributable risk of atopy. METHODS: UCB was collected at birth and maternal atopic status confirmed by skin prick testing (SPT) (n5 28 atopic, n5 18 non-atopic). Mononuclear cells (MNCs) were isolated from the UCB samples, cryopreserved, then later thawed and depleted of adherent cells. NAMNCs were incubated with human recombinant interleukin 5 (rhIL-5; 1ng/mL) and collected at 0, 24, 48 and 72 hours post-stimulation. Multiplex qPCR was performed to measure GATA1 and PRG2mRNA expression at all time points. RESULTS: Stimulation of cord blood NAMNCs with IL-5 resulted in steady downregulation of GATA1 expression beginning at 24 hours. Conversely, there was an upregulation of the PRG2 gene expression, as previously demonstrated. However, there were no significant differences in the GATA1 and PRG2 gene expression patterns of UCB samples from infants born to atopic mothers vs. non-atopics. (GATA1 P 5 0.2285, 0.3166, and 0.1174 24h, 48h and 72h, respectively; PRG2P 5 0.1663, 0.6285, and 0.7612 at 24h, 48h and 72h). CONCLUSIONS: In this study, multiplexed qPCR analysis of GATA1 and PRG2 mRNA expression showed no significant differences between infants born to atopic versus non-atopic mothers, but enhanced understanding of eosinophilopoesis in UCB.
197
Mast Cells Drive Tissue Inflammation By Producing IL-33 That Orchestrates a Unique Basophil Phenotype Dr. Chia-Lin Hsu, PhD, Dr. Paul Bryce, PhD; Division of Allergy-Immunology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL. RATIONALE: Skin, an important environmental interface, protects against allergens that can trigger local or systemic anaphylactic reactions in allergic individuals. Skin-resident mast cells drive an early edema followed by a late antigen-driven tissue inflammation (ADTI) characterized by inflammatory cell recruitment. While histamine is important for the immediate edema after encountering the antigens, the mechanisms that control the inflammatory cell influx are still unclear. METHODS: A passive cutaneous model was used to study the ADTI. Mice were intradermally injected with PBS in one ear and DNP-specific IgE in the other followed by intravenous challenge with DNP-HSA. Ear swelling was measured and cell infiltration was determined by flow cytometry. Murine bone marrow-derived basophils (BMBs) were generated in vitro by culturing with IL-3 and activated with IgE/DNP or IL-33. RESULTS: In vitro, we showed that IgE/DNP primed BMBs to produce Th2-associated cytokines like IL-4 and CCL24, while IL-33 activated basophils to secrete neutrophil-recruiting chemokines, such as IL-6, CXCL1 and CXCL2. In vivo, we showed that mast cell-derived IL-33 was sufficient and necessary to induce the late phase ear swelling. ST2 and IL-6-expressing basophils were also required for inflammatory cell recruitment. Furthermore, it was H4R responsible for basophil recruitment. CONCLUSIONS: Our finding indicated that mast cells acted as local sensors in responding to foreign antigens. Histamine was produced immediately and basophils were recruited through H4R. Migrated basophils were then activated by mast cell-derived IL-33 through ST2 and produced IL-6 to promote antigen-driven tissue inflammation by recruiting CD45+Gr-1high neutrophils.
SATURDAY
Abstracts AB55
J ALLERGY CLIN IMMUNOL VOLUME 133, NUMBER 2