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Flt3 Ligand (Flt3-L) Regulates Migratory Pattern of Lung Dendritic Cells in a Mouse Model of Allergic Airway Inflammation
S222 Abstracts
855
Identification of a Transcriptional Signature Associated with Specific Immune Response to Cockroach Allergen C. Cheadle1, P. S. Gao1, D. Grigoryev1, N. M. Rafaels1, J. M. Wright1, B. A. Plunkett1, Y. C. Chen1, M. Stockton1, A. Togias1, T. H. Beaty2, R. A. Mathias3, J. T. Schroeder1, K. C. Barnes1; 1Johns Hopkins School of Medicine, Baltimore, MD, 2Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, 3National Human Genome Research Institute, NIH, Baltimore, MD. RATIONALE: Sensitization and exposure to cockroach allergen is one of the strongest predictors of asthma morbidity, especially among African Americans. Our aims were to determine the molecular and immunological basis of cockroach sensitization and the specific response to cockroach antigen. METHODS: We investigated the effect of cockroach antigen on dendritic cell (DC) function and the ‘‘transcript signature’’ of the immune response to cockroach antigen with the use of high-throughput expression profiling of co-cultured plasmacytoid DCs (pDCs) and CD41 T cells. RESULTS: We observed significantly elevated levels of IL-13, IL-10 and TNF-a, but undetectable levels of IL-12p70, when cultures were exposed to cockroach antigen. A significant difference was observed for IL-13 between cockroach allergic and non-allergic individuals (p 5 0.039). Microarray analyses demonstrated a greater response at 48 hours compared to 4 hours, with 50 genes being uniquely expressed in cockroach antigentreated cells from cockroach allergic individuals, including CD14, S100A8, CCL8, and IFI44L. Because of the strong effect on the CD14 gene expression, we also conducted an association study between CD14 -260C/T genotypes and cockroach allergy among 595 atopic African Americans, but found no relations. However, in the in vitro study, CD14 gene expression was significantly increased upon cockroach exposure only in the CC genotype. Functional genomic analyses of these differentially expressed genes suggested the interferon signaling pathway as the most significant canonical pathway. CONCLUSIONS: We have identified differentially expressed genes and genes in the IFN-signaling pathway which should be further investigated for their role in the immune response to cockroach and in allergic cockroach sensitization.
856
TUESDAY
Peanut Extract Acts as a Th2-skewing Adjuvant B. Ruiter, H. A. Sampson, W. G. Shreffler; Mount Sinai School of Medicine, New York, NY. RATIONALE: Peanut allergy presents with relatively severe symptoms and is seldom outgrown, suggesting that peanut proteins are more allergenic than most other food proteins. Our objective in the current study was to test whether peanut allergens have Th2-polarizing effects on peripheral human myeloid DCs. METHODS: Myeloid DCs (CD11c1 BDCA11) and naive Th cells (CD41 CD45RA1) were isolated from buffy coats of healthy blood donors. DCs were incubated for 6 h with either medium alone, purified Arah1 or Arah2, peanut extract (PE), lipopolysaccharide (LPS) or cholera toxin (CT). Autologous naive T cells were then added in a 1:10 ratio of DC/ T cells. Low levels of staphylococcal enterotoxin B (0.01-0.1 pg/ml) were added to all cultures, to induce T cell responses. Cytokines were measured in culture supernatants after 5 days. RESULTS: Naive T cells incubated with PE-treated DCs produced significantly more IL-13 than T cells incubated with unstimulated DCs (4.2 fold increase; p < 0.001). Arah1-treated DCs also had this effect (2.47 fold; p < 0.01), but Arah2 did not (0.7 fold; NS). The ratio of the fold increase of IL13 to IFN-g production from T cells stimulated by PE-, Arah1- or Arah2treated DCs was 2.5, 1.7 and 0.68 respectively. Levels of IL-5 tended to be higher in T cells incubated with PE-treated (p 5 0.03) and Arah1-treated (p 5 0.18) DCs. LPS and CT controls induced strong Th1- and Th2-skewing, respectively. CONCLUSIONS: PE acts as a Th2-skewing adjuvant. Arah1, but not Arah2, appears to play an important role in this effect. The innate immune response to PE may be a decisive factor causing its allergenicity.
J ALLERGY CLIN IMMUNOL FEBRUARY 2009
857
Flt3 Ligand (Flt3-L) Regulates Migratory Pattern of Lung Dendritic Cells in a Mouse Model of Allergic Airway Inflammation Z. Shao, MD,, D. K. AgrawalPhD; Creighton University School of Medicine, Omaha, NE. RATIONALE: Flt3-L enhances the generation and recruitment of Th2suppressive lung CD11chiCD11blow DCs and decrease immunogenic CD11cloCD11bhi DCs. In this study, the migratory pattern of the lung dendritic cells under the influence of Flt3-L was examined. METHODS: Lung CD11chiCD11blow DCs and lung CD11cloCD11bhi DCs from PBS-treated, OVA-sensitized and OVA-sensitized-Flt3-L-treated mice were sorted by MACS and FACS, and labeled with fluorochrome-conjugated CD11c, CD11b, CCR2, CCR5 and CCR7 antibodies for flow cytometry analysis. The expression of CCL19 and CCL21 in thoracic lymph nodes was determined by IHC. Migratory pattern of the two lung DC subsets in response to CCL19 and CCL21 was examined in vitro using TransWell. The phenotype of thoracic lymph node cells was studied. RESULTS: CD11cloCD11bhi DCs had higher expression of CCR5, CCR7 and lower CCR2 expression than CD11chiCD11blo DCs. Flt3-L increased the expression of CCR2 in CD11chiCD11blo DCs and decreased CCR7 expression in CD11cloCD11bhi DCs in OVA-sensitized and challenged mice. A lower expression of CCL19 and CCL21 was observed in Flt3-L-treated mice than OVA-sensitized mice. CD11cloCD11bhi DCs demonstrated better migratory capacity than CD11chiCD11blo DCs in response to CCL19 and CCL21 but theirs migration was largely impaired in Flt3-L-treated-mice, while the CD11chiCD11blow DCs demonstrated stronger response to CCL2 and this was further enhanced in Flt3-L-treated mice. DCs in thoracic lymph nodes in OVA-sensitized mice had CD11clowCD11bhigh phenotype. CONCLUSIONS: Flt3-L regulates the recruitment and migration of lung DCs by modifying the expression of CCRs and their ligands recruiting more Th2-suppressive DCs in the lung and limiting the migration of immunogenic DCs to lymph nodes.
858
TLR4 Expression and Function in Children with Milk Allergy Y. Levy1,2, I. Lagovsky3, A. Vanichkin3, B. Garty1,2; 1 Schneider Children’s Medical Center of Israel, Petach Tikva, Israel, 2 Sackler Faculty of Medicine, Tel Aviv University, Tel -Aviv, Israel, 3Felsenstein Medical Research Center, Sackler Faculty of Medicine, Tel-Aviv University, Beilinson Campus, Petach Tikva, Israel. RATIONALE: The prevalence of milk allergy including fatal reactions has increased. Variations in innate immunity responses may predispose to development of allergic diseases. Little is known about TLR4 expression and function in food allergic humans. METHODS: Peripheral blood mononuclear cells of infants and children with documented IgE mediated milk allergy (n 5 13) and non allergic controls(n 5 5) were separated and cultured for 24 hours either alone or with LPS 1mg/ml and b casein 80mg/ml . The supernatants were tested for IL-6 and TNF-a levels using commercial ELISA kits;results were expressed as Mean 6 SEM (p value< 0.05 was considered statistically significant). For TLR4 membrane expression assay in FACS, adherent cells were recovered, and CD141 cells were stained with phycoerythrin conjugated anti human TLR4 mAb’s; the results were expressed as relative mean fluorescence intensity (rMFI). RESULTS: The rMFI of TLR4 membrane expression in monocytes of milk allergic patients in response to LPS and casein stimulation were 1.34 6 0.08 and 1.78 6 0.19 compared to controls: 1.68 6 0.18 and 1.42 6 0.05 respectively (p value-NS). IL-6 levels (pg/ml) in response to LPS and b casein stimulation were 22900 6 2776 and 4513 6 803 in allergic patients, and 14311 6 2021 and 464 6 18 in controls respectively (p value 5 0.02 and 0.003 respectively). TNF-a levels (pg/ml) in response to LPS and b casein were 2089 6 424 and 688 6 345 in allergic patients, and 1348 6 185 and 180 6 65 in controls respectively (p value-NS). CONCLUSIONS: Upon TLR4 ligand stimulation, children with milk allergy exhibit similar TLR4 membrane expression compared to non allergic controls.TLR4 mediated proinflammatory responses are not impaired, since TNF-a production is similar and IL-6 production is increased.
855
Identification of a Transcriptional Signature Associated with Specific Immune Response to Cockroach Allergen C. Cheadle1, P. S. Gao1, D. Grigoryev1, N. M. Rafaels1, J. M. Wright1, B. A. Plunkett1, Y. C. Chen1, M. Stockton1, A. Togias1, T. H. Beaty2, R. A. Mathias3, J. T. Schroeder1, K. C. Barnes1; 1Johns Hopkins School of Medicine, Baltimore, MD, 2Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, 3National Human Genome Research Institute, NIH, Baltimore, MD. RATIONALE: Sensitization and exposure to cockroach allergen is one of the strongest predictors of asthma morbidity, especially among African Americans. Our aims were to determine the molecular and immunological basis of cockroach sensitization and the specific response to cockroach antigen. METHODS: We investigated the effect of cockroach antigen on dendritic cell (DC) function and the ‘‘transcript signature’’ of the immune response to cockroach antigen with the use of high-throughput expression profiling of co-cultured plasmacytoid DCs (pDCs) and CD41 T cells. RESULTS: We observed significantly elevated levels of IL-13, IL-10 and TNF-a, but undetectable levels of IL-12p70, when cultures were exposed to cockroach antigen. A significant difference was observed for IL-13 between cockroach allergic and non-allergic individuals (p 5 0.039). Microarray analyses demonstrated a greater response at 48 hours compared to 4 hours, with 50 genes being uniquely expressed in cockroach antigentreated cells from cockroach allergic individuals, including CD14, S100A8, CCL8, and IFI44L. Because of the strong effect on the CD14 gene expression, we also conducted an association study between CD14 -260C/T genotypes and cockroach allergy among 595 atopic African Americans, but found no relations. However, in the in vitro study, CD14 gene expression was significantly increased upon cockroach exposure only in the CC genotype. Functional genomic analyses of these differentially expressed genes suggested the interferon signaling pathway as the most significant canonical pathway. CONCLUSIONS: We have identified differentially expressed genes and genes in the IFN-signaling pathway which should be further investigated for their role in the immune response to cockroach and in allergic cockroach sensitization.
856
TUESDAY
Peanut Extract Acts as a Th2-skewing Adjuvant B. Ruiter, H. A. Sampson, W. G. Shreffler; Mount Sinai School of Medicine, New York, NY. RATIONALE: Peanut allergy presents with relatively severe symptoms and is seldom outgrown, suggesting that peanut proteins are more allergenic than most other food proteins. Our objective in the current study was to test whether peanut allergens have Th2-polarizing effects on peripheral human myeloid DCs. METHODS: Myeloid DCs (CD11c1 BDCA11) and naive Th cells (CD41 CD45RA1) were isolated from buffy coats of healthy blood donors. DCs were incubated for 6 h with either medium alone, purified Arah1 or Arah2, peanut extract (PE), lipopolysaccharide (LPS) or cholera toxin (CT). Autologous naive T cells were then added in a 1:10 ratio of DC/ T cells. Low levels of staphylococcal enterotoxin B (0.01-0.1 pg/ml) were added to all cultures, to induce T cell responses. Cytokines were measured in culture supernatants after 5 days. RESULTS: Naive T cells incubated with PE-treated DCs produced significantly more IL-13 than T cells incubated with unstimulated DCs (4.2 fold increase; p < 0.001). Arah1-treated DCs also had this effect (2.47 fold; p < 0.01), but Arah2 did not (0.7 fold; NS). The ratio of the fold increase of IL13 to IFN-g production from T cells stimulated by PE-, Arah1- or Arah2treated DCs was 2.5, 1.7 and 0.68 respectively. Levels of IL-5 tended to be higher in T cells incubated with PE-treated (p 5 0.03) and Arah1-treated (p 5 0.18) DCs. LPS and CT controls induced strong Th1- and Th2-skewing, respectively. CONCLUSIONS: PE acts as a Th2-skewing adjuvant. Arah1, but not Arah2, appears to play an important role in this effect. The innate immune response to PE may be a decisive factor causing its allergenicity.
J ALLERGY CLIN IMMUNOL FEBRUARY 2009
857
Flt3 Ligand (Flt3-L) Regulates Migratory Pattern of Lung Dendritic Cells in a Mouse Model of Allergic Airway Inflammation Z. Shao, MD,, D. K. AgrawalPhD; Creighton University School of Medicine, Omaha, NE. RATIONALE: Flt3-L enhances the generation and recruitment of Th2suppressive lung CD11chiCD11blow DCs and decrease immunogenic CD11cloCD11bhi DCs. In this study, the migratory pattern of the lung dendritic cells under the influence of Flt3-L was examined. METHODS: Lung CD11chiCD11blow DCs and lung CD11cloCD11bhi DCs from PBS-treated, OVA-sensitized and OVA-sensitized-Flt3-L-treated mice were sorted by MACS and FACS, and labeled with fluorochrome-conjugated CD11c, CD11b, CCR2, CCR5 and CCR7 antibodies for flow cytometry analysis. The expression of CCL19 and CCL21 in thoracic lymph nodes was determined by IHC. Migratory pattern of the two lung DC subsets in response to CCL19 and CCL21 was examined in vitro using TransWell. The phenotype of thoracic lymph node cells was studied. RESULTS: CD11cloCD11bhi DCs had higher expression of CCR5, CCR7 and lower CCR2 expression than CD11chiCD11blo DCs. Flt3-L increased the expression of CCR2 in CD11chiCD11blo DCs and decreased CCR7 expression in CD11cloCD11bhi DCs in OVA-sensitized and challenged mice. A lower expression of CCL19 and CCL21 was observed in Flt3-L-treated mice than OVA-sensitized mice. CD11cloCD11bhi DCs demonstrated better migratory capacity than CD11chiCD11blo DCs in response to CCL19 and CCL21 but theirs migration was largely impaired in Flt3-L-treated-mice, while the CD11chiCD11blow DCs demonstrated stronger response to CCL2 and this was further enhanced in Flt3-L-treated mice. DCs in thoracic lymph nodes in OVA-sensitized mice had CD11clowCD11bhigh phenotype. CONCLUSIONS: Flt3-L regulates the recruitment and migration of lung DCs by modifying the expression of CCRs and their ligands recruiting more Th2-suppressive DCs in the lung and limiting the migration of immunogenic DCs to lymph nodes.
858
TLR4 Expression and Function in Children with Milk Allergy Y. Levy1,2, I. Lagovsky3, A. Vanichkin3, B. Garty1,2; 1 Schneider Children’s Medical Center of Israel, Petach Tikva, Israel, 2 Sackler Faculty of Medicine, Tel Aviv University, Tel -Aviv, Israel, 3Felsenstein Medical Research Center, Sackler Faculty of Medicine, Tel-Aviv University, Beilinson Campus, Petach Tikva, Israel. RATIONALE: The prevalence of milk allergy including fatal reactions has increased. Variations in innate immunity responses may predispose to development of allergic diseases. Little is known about TLR4 expression and function in food allergic humans. METHODS: Peripheral blood mononuclear cells of infants and children with documented IgE mediated milk allergy (n 5 13) and non allergic controls(n 5 5) were separated and cultured for 24 hours either alone or with LPS 1mg/ml and b casein 80mg/ml . The supernatants were tested for IL-6 and TNF-a levels using commercial ELISA kits;results were expressed as Mean 6 SEM (p value< 0.05 was considered statistically significant). For TLR4 membrane expression assay in FACS, adherent cells were recovered, and CD141 cells were stained with phycoerythrin conjugated anti human TLR4 mAb’s; the results were expressed as relative mean fluorescence intensity (rMFI). RESULTS: The rMFI of TLR4 membrane expression in monocytes of milk allergic patients in response to LPS and casein stimulation were 1.34 6 0.08 and 1.78 6 0.19 compared to controls: 1.68 6 0.18 and 1.42 6 0.05 respectively (p value-NS). IL-6 levels (pg/ml) in response to LPS and b casein stimulation were 22900 6 2776 and 4513 6 803 in allergic patients, and 14311 6 2021 and 464 6 18 in controls respectively (p value 5 0.02 and 0.003 respectively). TNF-a levels (pg/ml) in response to LPS and b casein were 2089 6 424 and 688 6 345 in allergic patients, and 1348 6 185 and 180 6 65 in controls respectively (p value-NS). CONCLUSIONS: Upon TLR4 ligand stimulation, children with milk allergy exhibit similar TLR4 membrane expression compared to non allergic controls.TLR4 mediated proinflammatory responses are not impaired, since TNF-a production is similar and IL-6 production is increased.