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Atrial Natriuretic Peptide (ANP)-mediated Maturation and Cytokine Phenotype of Human Dendritic Cells (DCs) Involve Demethylation of the SOCS3 Promoter
RESULTS: The change in paw swelling over time was increased for GRK6 deficient and GRK2 heterozygote mice compared to controls (GRK6KO p50.0004 slope, p50.0002 curvature; GRK2(1,-), p50.0043 slope, p50.0052 curvature). Clinical disease index was also worsened compared to controls (GRK6KO p50.0034 slope, p50.0018 curvature; GRK2(1,-), p50.0099 slope, p50.0061 curvature). In vitro granulocyte chemotaxis toward LTB4 and C5a was increased in GRK6 and GRK2, but not GRK5, deficient mice compared to controls. Generalized systemic inflammation was uniquely observed in GRK6 deficient mice, which had enhanced weight loss (p0.05 Days 3-10) and increased systemic production of IL-6 and IL-1b. CONCLUSIONS: The GRK system is an important regulatory pathway in the K/BxN serum transfer model of inflammatory arthritis, but differs between the GRK subtyptes. Deficiency of GRK6 and GRK2, but not GRK5, enhances the chemotaxis of granulocytes, which may lead to worsening arthritis. Funding: K08 AI070684-01, K12 HD01441, ACR-REF Physician Scientist Award
of TSLP and results in recruitment of TSLPR bearing cells in the cutaneous allergen-induced LPR in atopic subjects. METHODS: Skin biopsies were obtained from atopic subjects (n 5 9) at various times after cutaneous allergen challenge. In situ hybridization and immunohistochemistry were used to determine TSLP mRNA expression and to measure infiltration of TSLPR1/CD11c1 DC in skin LPR. RTPCR and flow cytometry were employed to analyze TSLPR expression on isolated myeloid DCs. RESULTS: Allergen-induced skin TSLP expression occurred as early as 1 hour after allergen challenge, whereas TSLPR1 and CD11c1 cells infiltrated relatively late (24-48 hrs). The majority of TSLPR1 cells were CD11c1 DCs. Freshly isolated blood myeloid DCs expressed both TSLPR and IL-7Ra chains. Maturation and stimulation with TSLP or Poly (I:C) in vitro upregulated the expression of both TSLPR and IL-7Ra chains in DCs but not in CRTH21CD41 T cells. CONCLUSIONS: Our data suggest that TSLP plays a role in augmenting, through DC recruitment and activation, the development of Th2-type T cells in allergic inflammation.
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Flt3 Ligand (FL) Treatment Generates Multiple Subsets of Myeloid Dendritic Cells (DCs) with Differential Phenotypic and Functional Properties in Mouse Model of Asthma A. S. Bharadwaj, D. K. Agrawal; Creighton University, Omaha, NE. RATIONALE: We observed an increase in the number of myeloid DCs after FL in a mouse model of asthma. Contrary to popular belief, these myeloid DCs showed decreased ability to stimulate T cells. Therefore, we examined if FL generates more than one population of myeloid DCs. METHODS: Female Balb/c mice were sensitized and challenged with ovalbumin. Mice with established airway hyperresponsiveness to methacholine were administered with rmFL (10 mg/mice/day). After 10 days of treatment, lungs and spleen were collected and CD11c1 cells sorted using Magnetic assisted cell sorter (MACS). CD11c1 cells were further labeled with fluorochrome-tagged antibodies and sorted using the FACS Aria. T cell proliferation in response to individual cell type was examined and expression of PDL1 and PDL2 genes was detected. RESULTS: Two populations (CD11c1CD11blo, CD11chiCD11b1) of myeloid DCs were observed in the spleen and three populations (CD11c1CD11blo, CD11chiCD11bint, CD11c1CD11bhi) in the lungs. FL did not alter the myeloid DC subsets of the spleen. However, lung DCs showed differential expression of not only CD11c and CD11b but also MHCII and B220. The relative expression of B220 and MHCII on the three populations of lung myeloid DCs correlated with their ability to stimulate T cell proliferation. Expression of PDL1 and PDL2 was higher in the lung DC population from FL-treated mice expressing CD11c1CD11bhi and this correlated with decreased T cell proliferation. CONCLUSION: These data suggests that Flt3 ligand generates multiple subsets of myeloid DCs, one of which is involved in the suppression of T cell proliferation via the PD1-PDL pathway. (Supported by NIH grant HL070885) Funding: NIH grant HL070885
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Thymic Stromal Lymphopoietin (TSLP) and Receptor (TSLPR) and CD11c1 Dendritic Cells (DC) in Allergen-induced Cutaneous Late Phase Responses (LPR) in Atopic Subjects S. Ying1, Y. H. Wang2, Y. J. Liu2, Q. Meng1, A. B. Kay3, S. Phipps3, T. H. Lee1, C. J. Corrigan1; 1King’s College London, London, UNITED KINGDOM, 2Centre of Cancer Immunolgy, MD Anderson Cancer Centre, Houston, TX, 3Imperial College, London, UNITED KINGDOM. RATIONALE: TSLP is an IL-7-like cytokine that triggers dendritic cellmediated T helper (Th)2 inflammatory responses through a receptor consisting of a heterodimer of the IL-7 receptor alpha (IL-7Ra) chain and the TSLP receptor (TSLPR) which resembles the cytokine receptor common gamma chain. Dendritic cells activated by TSLP prime development of CD41 T cells into Th2 cells contributing to the pathogenesis of allergic inflammation. We hypothesized that allergen exposure induces expression
Atrial Natriuretic Peptide (ANP)-mediated Maturation and Cytokine Phenotype of Human Dendritic Cells (DCs) Involve Demethylation of the SOCS3 Promoter W. Zhang, X. Cao, G. Hellermann, R. F. Lockey, S. S. Mohapatra; USF, Tampa, FL. RATIONALE: ANP modulates the maturation and cytokine phenotype of DCs, particularly by altering their IL-10 production, but the mechanism is unclear. Since, the suppressor of cytokine signaling-3, SOCS3, inhibits the activity of DCs, the action of SOCS3 on ANP-activated DCs was studied by demethylation of the SOCS3 promoter. METHODS: Transfected DCs were treated with the demethylating reagent 59-aza-29-deoxycytidine (59-aza-29dC), SOCS3 expression was determined by immunoblot or RT-PCR, and supernatant cytokines were measured by Bio-Plex array. To assess the effect of methylation on SOCS3 expression, a SOCS3-P-luciferase reporter plasmid was methylated and co-transfected into DCs with pANP. Luciferase activity was measured 48 hours after transfection. The maturation of DCs was assessed using the FITC-dextran phagocytosis assay. RESULTS: ANP downregulated SOCS3 expression compared to control, but SOCS3 was up-regulated by 59-aza-29dC demethylation. Also, treatment of pANP-transfected DCs with siRNA to SOCS3 leads to the upregulation of IL-10 and IFN-g. Moreover, demethylation of CpG islands in the SOCS3 promoter elevates the expression of SOCS3 and enhances maturation of human DCs. CONCLUSIONS: These results indicate that ANP mediated maturation of DCs involves demethylation of the SOCS3 promoter.
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Abstracts S235
J ALLERGY CLIN IMMUNOL VOLUME 119, NUMBER 1
of TSLP and results in recruitment of TSLPR bearing cells in the cutaneous allergen-induced LPR in atopic subjects. METHODS: Skin biopsies were obtained from atopic subjects (n 5 9) at various times after cutaneous allergen challenge. In situ hybridization and immunohistochemistry were used to determine TSLP mRNA expression and to measure infiltration of TSLPR1/CD11c1 DC in skin LPR. RTPCR and flow cytometry were employed to analyze TSLPR expression on isolated myeloid DCs. RESULTS: Allergen-induced skin TSLP expression occurred as early as 1 hour after allergen challenge, whereas TSLPR1 and CD11c1 cells infiltrated relatively late (24-48 hrs). The majority of TSLPR1 cells were CD11c1 DCs. Freshly isolated blood myeloid DCs expressed both TSLPR and IL-7Ra chains. Maturation and stimulation with TSLP or Poly (I:C) in vitro upregulated the expression of both TSLPR and IL-7Ra chains in DCs but not in CRTH21CD41 T cells. CONCLUSIONS: Our data suggest that TSLP plays a role in augmenting, through DC recruitment and activation, the development of Th2-type T cells in allergic inflammation.
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Flt3 Ligand (FL) Treatment Generates Multiple Subsets of Myeloid Dendritic Cells (DCs) with Differential Phenotypic and Functional Properties in Mouse Model of Asthma A. S. Bharadwaj, D. K. Agrawal; Creighton University, Omaha, NE. RATIONALE: We observed an increase in the number of myeloid DCs after FL in a mouse model of asthma. Contrary to popular belief, these myeloid DCs showed decreased ability to stimulate T cells. Therefore, we examined if FL generates more than one population of myeloid DCs. METHODS: Female Balb/c mice were sensitized and challenged with ovalbumin. Mice with established airway hyperresponsiveness to methacholine were administered with rmFL (10 mg/mice/day). After 10 days of treatment, lungs and spleen were collected and CD11c1 cells sorted using Magnetic assisted cell sorter (MACS). CD11c1 cells were further labeled with fluorochrome-tagged antibodies and sorted using the FACS Aria. T cell proliferation in response to individual cell type was examined and expression of PDL1 and PDL2 genes was detected. RESULTS: Two populations (CD11c1CD11blo, CD11chiCD11b1) of myeloid DCs were observed in the spleen and three populations (CD11c1CD11blo, CD11chiCD11bint, CD11c1CD11bhi) in the lungs. FL did not alter the myeloid DC subsets of the spleen. However, lung DCs showed differential expression of not only CD11c and CD11b but also MHCII and B220. The relative expression of B220 and MHCII on the three populations of lung myeloid DCs correlated with their ability to stimulate T cell proliferation. Expression of PDL1 and PDL2 was higher in the lung DC population from FL-treated mice expressing CD11c1CD11bhi and this correlated with decreased T cell proliferation. CONCLUSION: These data suggests that Flt3 ligand generates multiple subsets of myeloid DCs, one of which is involved in the suppression of T cell proliferation via the PD1-PDL pathway. (Supported by NIH grant HL070885) Funding: NIH grant HL070885
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Thymic Stromal Lymphopoietin (TSLP) and Receptor (TSLPR) and CD11c1 Dendritic Cells (DC) in Allergen-induced Cutaneous Late Phase Responses (LPR) in Atopic Subjects S. Ying1, Y. H. Wang2, Y. J. Liu2, Q. Meng1, A. B. Kay3, S. Phipps3, T. H. Lee1, C. J. Corrigan1; 1King’s College London, London, UNITED KINGDOM, 2Centre of Cancer Immunolgy, MD Anderson Cancer Centre, Houston, TX, 3Imperial College, London, UNITED KINGDOM. RATIONALE: TSLP is an IL-7-like cytokine that triggers dendritic cellmediated T helper (Th)2 inflammatory responses through a receptor consisting of a heterodimer of the IL-7 receptor alpha (IL-7Ra) chain and the TSLP receptor (TSLPR) which resembles the cytokine receptor common gamma chain. Dendritic cells activated by TSLP prime development of CD41 T cells into Th2 cells contributing to the pathogenesis of allergic inflammation. We hypothesized that allergen exposure induces expression
Atrial Natriuretic Peptide (ANP)-mediated Maturation and Cytokine Phenotype of Human Dendritic Cells (DCs) Involve Demethylation of the SOCS3 Promoter W. Zhang, X. Cao, G. Hellermann, R. F. Lockey, S. S. Mohapatra; USF, Tampa, FL. RATIONALE: ANP modulates the maturation and cytokine phenotype of DCs, particularly by altering their IL-10 production, but the mechanism is unclear. Since, the suppressor of cytokine signaling-3, SOCS3, inhibits the activity of DCs, the action of SOCS3 on ANP-activated DCs was studied by demethylation of the SOCS3 promoter. METHODS: Transfected DCs were treated with the demethylating reagent 59-aza-29-deoxycytidine (59-aza-29dC), SOCS3 expression was determined by immunoblot or RT-PCR, and supernatant cytokines were measured by Bio-Plex array. To assess the effect of methylation on SOCS3 expression, a SOCS3-P-luciferase reporter plasmid was methylated and co-transfected into DCs with pANP. Luciferase activity was measured 48 hours after transfection. The maturation of DCs was assessed using the FITC-dextran phagocytosis assay. RESULTS: ANP downregulated SOCS3 expression compared to control, but SOCS3 was up-regulated by 59-aza-29dC demethylation. Also, treatment of pANP-transfected DCs with siRNA to SOCS3 leads to the upregulation of IL-10 and IFN-g. Moreover, demethylation of CpG islands in the SOCS3 promoter elevates the expression of SOCS3 and enhances maturation of human DCs. CONCLUSIONS: These results indicate that ANP mediated maturation of DCs involves demethylation of the SOCS3 promoter.
MONDAY
Abstracts S235
J ALLERGY CLIN IMMUNOL VOLUME 119, NUMBER 1