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C and DNA Fragmentation
Abstracts S269
J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 2
Eosinophils Are Not Involved in the Sensitization Phase of Allergic Responses T. Kobayashi, K. Iijima, J. L. Checkel, H. Kita; Mayo Clinic Rochester, Rochester, MN. RATIONALE: Eosinophils and basophils have been implicated in allergic responses. During the effector phase, eosinophils may play roles in Th2 cell recruitment by modulating chemokine production. Basophils may play roles in initiating Th2 responses in animals exposed to protease(s). We used a heterologous sensitization model to investigate the roles of eosinophils in allergen sensitization. METHODS: Wild-type BALB/cJ (WT) and eosinophil-deficient double GATA-1 knockout (C.Cg-Gata1tm6Sho/J, DdblGATA) mice were exposed intranasally to an airway Th2 adjuvant, Alternaria alternata (ALT) extract, and a model allergen, ovalbumin (OVA). Serum levels of antibodies and splenocyte recall responses to OVA were examined. Airway inflammation was determined in mice challenged intranasally with OVA. RESULTS: Airway exposure of naı¨ve WT mice to OVA alone did not induce immune responses to OVA. In contrast, airway exposure of WT mice to OVA in the presence of ALT induced marked increases in total IgE and OVA-specific IgG1 and also airway and splenocyte Th2-type recall responses to OVA. In DdblGATA mice, airway exposure to OVA with ALT induced robust Th2-type antibody responses and splenocyte recall responses to OVA; the responses of DdblGATA mice were comparable to or higher than those of WT mice. Furthermore, airway OVA challenge of DdblGATA mice, which were previously exposed to OVA with ALT, induced airway lymphocyte recruitment and increased airway levels of IL-4, IL-5 and IL-13. CONCLUSIONS: Unlike basophils, eosinophils may not be involved in development of adaptive Th2 responses. Functional dichotomies may exist between these cell types during the sensitization and effector phases of allergic responses.
1043
Polymers Containing 6'-sulfated Sialyl Lewis X (6'-susLex) Selectively Engage Siglec-8 on Human Eosinophils. S. A. Hudson1, N. Bovin2, P. R. Crocker3, B. S. Bochner1; 1Johns Hopkins Asthma and Allergy Center, Baltimore, MD, 2Russian Academy of Sciences, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russian Federation, 3Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, Dundee, United Kingdom. RATIONALE: Siglec-8 is a cell-surface protein with lectin activity that is selectively expressed by eosinophils and mast cells. It preferentially binds the glycan 6’-su-sLeX. Engagement of Siglec-8 with specific antibodies results in eosinophil apoptosis and inhibition of FceRI-dependent mast cell mediator release. We determined whether a soluble, synthetic polyacrylamide polymer decorated with 6’-su-sLeX glycan selectively binds to Siglec-8. METHODS: Eosinophils were purified from blood using density gradient centrifugation and negative selection. HEK cells were stably transfected with wild type Siglec-8. Attachment to low (40 kDa) and high molecular weight (2000 kDa) biotinylated multivalent polyacrylamide polymers decorated with 6’-su-sLeX or structurally related control glycans (e.g., sLeX) was examined in static adhesion assays and by flow cytometry. RESULTS: HEK cell transfectants, but not eosinophils, bound the 40 kDa polymer. Both cell types bound the 2000 kDa polymer. Polymer binding was inhibited by polyclonal Siglec-8 antibody and neither cell type bound any of the control glycan polymers. In whole blood, eosinophils were the only cells to detectably bind the 2000 kDa polymer. HEK cell transfectants bound to plates coated with 6’-su-sLeX polymers but not to control glycan polymers. CONCLUSIONS: Antibodies as well as a large multivalent form of 6’-susLeX can be used to selectively engage Siglec-8. Additional studies will be needed to determine whether 6’-su-sLeX polymer or a related glycomimetic can reproduce the biology seen with Siglec-8 antibodies. If so, such agents might be useful for in vitro or in vivo manipulation of eosinophil and mast cell function.
1044
NO Induces Apoptosis of GM-CSF-treated Eosinophils via Mitochondria and Caspase-6-dependent Lamin A/C and DNA Fragmentation P. Ilmarinen1, E. Moilanen1, H. Kankaanranta1,2; 1Immunopharmacology Research Group, Medical School, University of Tampere and Tampere University Hospital, Tampere, Finland, 2Department of Respiratory Medicine, Seina¨joki Central Hospital, Seina¨joki, Finland. RATIONALE: Prolonged eosinophil survival is a critical factor leading to persistent eosinophilic lung inflammation in asthma. Nitric oxide (NO) was previously shown to regulate eosinophil apoptosis. Our aim was to clarify the signalling mechanisms leading to NO-induced apoptosis in granulocyte macrophage-colony stimulating factor (GM-CSF)-treated eosinophils. METHODS: Apoptosis of human peripheral blood eosinophils was determined by flow cytometric analysis of relative DNA content. Mitochondrial membrane potential was assessed by staining eosinophils with cationic mitochondrial dye JC-1 followed by flow cytometric analysis. Western blotting was used to study lamin A/C and caspase-6 proenzyme expression. RESULTS: NO-donor S-nitroso-N-acetyl-D, L-penicillamine (SNAP) prevented GM-CSF-afforded eosinophil survival by inducing DNA breakdown, indicative of apoptosis. NO-induced DNA breakdown in GM-CSF-treated cells was partly reversed by bongkrekic acid, an inhibitor of mitochondrial permeability transition pore (MPTP) opening. Furthermore, SNAP attenuated mitochondrial membrane potential and induced lamin A/C cleavage into a 28 kDa fragment. Western blotting analysis showed reduction in caspase-6 proenzyme expression after treatment with GM-CSF and SNAP indicative of caspase-6 activation. Additionally, caspase-6 inhibitor zVEID-fmk abrogated SNAP-induced DNA fragmentation and lamin A/C cleavage in GM-CSF-treated cells. CONCLUSIONS: Our results imply that NO induces apoptosis of GMCSF-treated human eosinophils through a pathway involving MPTP opening, loss of mitochondrial membrane potential and caspase-6-dependent lamin A/C cleavage and DNA fragmentation.
TUESDAY
1042
J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 2
Eosinophils Are Not Involved in the Sensitization Phase of Allergic Responses T. Kobayashi, K. Iijima, J. L. Checkel, H. Kita; Mayo Clinic Rochester, Rochester, MN. RATIONALE: Eosinophils and basophils have been implicated in allergic responses. During the effector phase, eosinophils may play roles in Th2 cell recruitment by modulating chemokine production. Basophils may play roles in initiating Th2 responses in animals exposed to protease(s). We used a heterologous sensitization model to investigate the roles of eosinophils in allergen sensitization. METHODS: Wild-type BALB/cJ (WT) and eosinophil-deficient double GATA-1 knockout (C.Cg-Gata1tm6Sho/J, DdblGATA) mice were exposed intranasally to an airway Th2 adjuvant, Alternaria alternata (ALT) extract, and a model allergen, ovalbumin (OVA). Serum levels of antibodies and splenocyte recall responses to OVA were examined. Airway inflammation was determined in mice challenged intranasally with OVA. RESULTS: Airway exposure of naı¨ve WT mice to OVA alone did not induce immune responses to OVA. In contrast, airway exposure of WT mice to OVA in the presence of ALT induced marked increases in total IgE and OVA-specific IgG1 and also airway and splenocyte Th2-type recall responses to OVA. In DdblGATA mice, airway exposure to OVA with ALT induced robust Th2-type antibody responses and splenocyte recall responses to OVA; the responses of DdblGATA mice were comparable to or higher than those of WT mice. Furthermore, airway OVA challenge of DdblGATA mice, which were previously exposed to OVA with ALT, induced airway lymphocyte recruitment and increased airway levels of IL-4, IL-5 and IL-13. CONCLUSIONS: Unlike basophils, eosinophils may not be involved in development of adaptive Th2 responses. Functional dichotomies may exist between these cell types during the sensitization and effector phases of allergic responses.
1043
Polymers Containing 6'-sulfated Sialyl Lewis X (6'-susLex) Selectively Engage Siglec-8 on Human Eosinophils. S. A. Hudson1, N. Bovin2, P. R. Crocker3, B. S. Bochner1; 1Johns Hopkins Asthma and Allergy Center, Baltimore, MD, 2Russian Academy of Sciences, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russian Federation, 3Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, Dundee, United Kingdom. RATIONALE: Siglec-8 is a cell-surface protein with lectin activity that is selectively expressed by eosinophils and mast cells. It preferentially binds the glycan 6’-su-sLeX. Engagement of Siglec-8 with specific antibodies results in eosinophil apoptosis and inhibition of FceRI-dependent mast cell mediator release. We determined whether a soluble, synthetic polyacrylamide polymer decorated with 6’-su-sLeX glycan selectively binds to Siglec-8. METHODS: Eosinophils were purified from blood using density gradient centrifugation and negative selection. HEK cells were stably transfected with wild type Siglec-8. Attachment to low (40 kDa) and high molecular weight (2000 kDa) biotinylated multivalent polyacrylamide polymers decorated with 6’-su-sLeX or structurally related control glycans (e.g., sLeX) was examined in static adhesion assays and by flow cytometry. RESULTS: HEK cell transfectants, but not eosinophils, bound the 40 kDa polymer. Both cell types bound the 2000 kDa polymer. Polymer binding was inhibited by polyclonal Siglec-8 antibody and neither cell type bound any of the control glycan polymers. In whole blood, eosinophils were the only cells to detectably bind the 2000 kDa polymer. HEK cell transfectants bound to plates coated with 6’-su-sLeX polymers but not to control glycan polymers. CONCLUSIONS: Antibodies as well as a large multivalent form of 6’-susLeX can be used to selectively engage Siglec-8. Additional studies will be needed to determine whether 6’-su-sLeX polymer or a related glycomimetic can reproduce the biology seen with Siglec-8 antibodies. If so, such agents might be useful for in vitro or in vivo manipulation of eosinophil and mast cell function.
1044
NO Induces Apoptosis of GM-CSF-treated Eosinophils via Mitochondria and Caspase-6-dependent Lamin A/C and DNA Fragmentation P. Ilmarinen1, E. Moilanen1, H. Kankaanranta1,2; 1Immunopharmacology Research Group, Medical School, University of Tampere and Tampere University Hospital, Tampere, Finland, 2Department of Respiratory Medicine, Seina¨joki Central Hospital, Seina¨joki, Finland. RATIONALE: Prolonged eosinophil survival is a critical factor leading to persistent eosinophilic lung inflammation in asthma. Nitric oxide (NO) was previously shown to regulate eosinophil apoptosis. Our aim was to clarify the signalling mechanisms leading to NO-induced apoptosis in granulocyte macrophage-colony stimulating factor (GM-CSF)-treated eosinophils. METHODS: Apoptosis of human peripheral blood eosinophils was determined by flow cytometric analysis of relative DNA content. Mitochondrial membrane potential was assessed by staining eosinophils with cationic mitochondrial dye JC-1 followed by flow cytometric analysis. Western blotting was used to study lamin A/C and caspase-6 proenzyme expression. RESULTS: NO-donor S-nitroso-N-acetyl-D, L-penicillamine (SNAP) prevented GM-CSF-afforded eosinophil survival by inducing DNA breakdown, indicative of apoptosis. NO-induced DNA breakdown in GM-CSF-treated cells was partly reversed by bongkrekic acid, an inhibitor of mitochondrial permeability transition pore (MPTP) opening. Furthermore, SNAP attenuated mitochondrial membrane potential and induced lamin A/C cleavage into a 28 kDa fragment. Western blotting analysis showed reduction in caspase-6 proenzyme expression after treatment with GM-CSF and SNAP indicative of caspase-6 activation. Additionally, caspase-6 inhibitor zVEID-fmk abrogated SNAP-induced DNA fragmentation and lamin A/C cleavage in GM-CSF-treated cells. CONCLUSIONS: Our results imply that NO induces apoptosis of GMCSF-treated human eosinophils through a pathway involving MPTP opening, loss of mitochondrial membrane potential and caspase-6-dependent lamin A/C cleavage and DNA fragmentation.
TUESDAY
1042