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Hypoxia Modulates Human Eosinophils Signalling and Function.
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Hypoxia Modulates Human Eosinophils Signalling and Function. A. H. Nissim Ben Efraim1, I. Puxeddu1, R. Eliashar2, F. Levi-Schaffer1; 1 The Hebrew University of Jerusalem, Jerusalem, Israel, 2Hadassah Medical Center, Jerusalem, Israel. RATIONALE: Eosinophils are involved in different inflammatory processes including allergic inflammation in which angiogenesis has been documented. Angiogenesis is probably connected to the hypoxia that characterizes inflamed tissues. Eosinophils produce VEGF and are pro-angiogenic. However, to the best of our knowledge, no study has been performed to verify the existence of a direct link between eosinophils, hypoxia and angiogenesis in allergic inflammation. METHODS: Human peripheral blood eosinophils were cultured in normoxic or hypoxic (<3% O2) conditions with or without GM-CSF (20ng/ ml). HIF-1a levels and MAPK phosphorylation were analyzed by immunoblot analysis. Angiogenic mediators release was evaluated by ELISA. RESULTS: Hypoxia increased VEGF release and HIF-1a, phosphoERK1/2 and phospho-p38 protein levels, particularly in the presence of GM-CSF. The ERK1/2 inhibitor PD98059 significantly decreased VEGF release. In the presence of GM-CSF in normoxia the inhibition decreased VEGF from 42 6 12.7 pg/ml to 22.9 6 9.97 pg/ml, and in hypoxia from 165.62 6 63.83 pg/ml to 23.77 6 6.18 pg/ml. Also with the p38 MAPK inhibitor SB203580 VEGF release decreased though not significantly. PD98059 caused HIF-1a expression decrease both in normoxia and in hypoxia, and in hypoxia it reached levels similar to the ones in normoxia. SB20358 decreased HIF-1a expression slightly but not significantly. CONCLUSIONS: Eosinophils respond to hypoxia by releasing increased amounts of pro-angiogenic factors and by up-regulating HIF-1alpha expression. This effect is augmented by the presence of GM-CSF and involves MAPK phosphorylation. These results are important for the understanding of eosinophils behaviour in inflamed tissue and for their role in angiogenesis.
189
Activation of Dendritic Cells by IL-33 Initiates a Th2 Response M. A. Rank, T. Kabayashi, K. R. Bartemes, H. Kita; Mayo Clinic, Rochester, MN. RATIONALE: IL-33, a new IL-1 family cytokine, is implicated in Th2type responses. However, the cellular targets for IL-33 are poorly understood. Because dendritic cells (DCs) are important in immune regulation, we hypothesized that DCs respond to IL-33 and trigger Th2 responses. METHODS: DCs were derived from murine bone marrow. Expression of the IL-33 receptor, ST2, was examined by FACS and real-time RT-PCR. After culturing DCs with IL-33 or LPS (as positive control) for 24 hours, activation of DCs was examined by FACS (MHC II and CD86 expression) and by ELISA (IL-6 and IL-12 production). The ability of IL-33-activated DCs to differentiate T cells was assessed by culturing DCs with naı¨ve CD4 1 T cells for 6 or 10 days and by measuring supernatant cytokines. RESULTS: ST2 mRNA was expressed abundantly in highly purified DCs, and ST2 protein was detected intracellularly by FACS. Incubation of DCs with IL-33 (as low as 1 ng/ml) increased MHC II and CD86 expression (p < 0.05) and induced IL-6 production (p < 0.01), but not IL-12. Anti-ST2 antibody inhibited IL-33-induced IL-6 production by approximately 60% (p < 0.03); LPS-induced IL-6 production was unaffected. Incubation of naive CD4 1 T cells, DCs, and IL-33 induced marked production of IL-5 and IL-13 (p < 0.05); IL-4 and IFN-g were undetectable. Incubation of DCs or naive CD4 1 T cells alone with IL-33 failed to stimulate IL-5 or IL-13 production. CONCLUSIONS: DCs respond to IL-33 through ST2. DCs plus IL-33 stimulate CD4 1 T cell production of Th2 cytokines. IL-33 and DCs may represent a new pathway to initiate Th2-type inflammation.
190
Topical N-acetyl cysteine (NAC) Reduces Late Phase Nasal Symptoms Following Ragweed Challenge C. J. Lane, D. R. Redding, K. A. Gonzalez, V. J. Cardenas, I. Boldogh, S. Sur; University of Texas Medical Branch, Galveston, TX. RATIONALE: Reactive oxygen species (ROS) generated by pollen NADPH oxidase augment aeroallergen induced airway inflammation. We examined the ability of the antioxidant NAC to modulate nasal symptoms following allergen challenge. METHODS: Ragweed sensitive patients were identified using skin prick testing and underwent nasal provocation challenges with either saline or ragweed extract (RWE.) Nasal symptom scores were recorded at baseline and at regular intervals following challenge. A late phase allergic response to RWE was defined as a difference of greater than or equal to 4 between the combined nasal symptom scores at 5 and 6 hours between RWE and saline challenge. Patients who demonstrated a late phase response to RWE then underwent challenge with RWE preceded by topical delivery of NAC in the nose. RESULTS: 3 of 8 subjects (38%) demonstrated a late phase reaction after RWE challenge and entered the open label NAC phase of the study. The total nasal symptom score (measured as change from baseline, mean 6 SEM) at 0.5 hours after allergen challenge was -1 6 0.6 for saline, 5.3 6 0.9 for RWE and 3.3 6 0.9 for NAC 1 RWE (p 5 NS vs RWE.) Scores at 5 hours were -1.3 6 0.9 for saline, 2.7 6 0.9 for RWE and -1 6 0.6 for NAC 1 RWE (p < 0.05 vs RWE.) Scores at 6 hours were -1.3 6 0.9 for saline, 2.7 6 0.9 for RWE and -1 6 1 for NAC 1 RWE (p < 0.05 vs RWE.) CONCLUSIONS: Topical NAC administered prior to RWE challenge reduced the late phase allergic response mediated nasal symptoms. Oxidative stress induced by RWE contributes to the nasal late phase response and may represent a therapeutic target for allergic airway disease.
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Comparison of Exhaled Breath Condensate and Sputum Induced by Hyperosmotic Saline Inhalation as Indicators of Airway Inflammation Associated with Bronchial Asthma T. Shimoda1, Y. Obase2, M. Imaoka1, R. Kishikawa1, S. Nishima1; 1Clinical Research Center, Fukuoka National Hospital, Fukuoka, Japan, 2Division of Respiratory Disease, Kawasaki Medical School, Kurashiki, Japan. RATIONALE: Testing of sputum induced by hypertonic saline inhalation has been established as a useful indicator of airway inflammation associated with bronchial asthma. Although exhaled breath condensate(EBC) can be collected simply and noninvasively, it is unclear whether it can serve as a valid indicator of airway inflammation. METHODS: The study involved 291 asthmatic patients who had not undergone steroid inhalation. First, each subject breathed at rest for 15 minutes using an ECoScreen (Jaeger, Germany), and EBC was collected. Then, each subject inhaled 3% hypertonic saline for 20 minutes, and sputum was collected, centrifuged, and its supernatant stored at -808C. Both samples from each subject were subsequently assayed for 20 substances by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: Assay of supernatant of induced sputum revealed the following substances: ECP (93.2 6 84.9ng/ml), TNF-a (62.5 6 96.5 pg/ml), IL5 (3.9 6 7.5 pg/ml), VEGF (227.9 6 243.5 pg/ml), MMP-9 (66.1 6 116.7ng/ml), IL-10 (31.4 6 26.2 pg/ml), IL-12 (27.0 6 30.2 pg/ml), IL13 (13.3 6 13.8 pg/ml), Histamine (16.1 6 21.4 nM/L), Fibronectin (1.04 6 1.19 mg/ml), LTE4 (691.7 6 367.1 pg/ml), and LTB4 (161.8 6 170.1 pg/ml). The levels of TGF-b, TIMP-1, IFN-g, GM-CSF, PDGF, FGF, IL-4 and Eotaxin were beneath the limits of detection. Assay of EBC revealed two substances, IL-13 (2.9 6 2.1 pg/ml) and LTE4 (175.7 6 221.6 pg/ml), while the other substances were beneath the limits of detection. CONCLUSIONS: Induced sputum is useful as an indicator of airway inflammation associated with bronchial asthma, since both the cellular component fraction and the supernatant of sputum can be evaluated. EBC, on the other hand, contains fewer quantifiable substances. The clinical usefulness of EBC thus appears to be quite limited.
SATURDAY
Abstracts S53
J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 2
Hypoxia Modulates Human Eosinophils Signalling and Function. A. H. Nissim Ben Efraim1, I. Puxeddu1, R. Eliashar2, F. Levi-Schaffer1; 1 The Hebrew University of Jerusalem, Jerusalem, Israel, 2Hadassah Medical Center, Jerusalem, Israel. RATIONALE: Eosinophils are involved in different inflammatory processes including allergic inflammation in which angiogenesis has been documented. Angiogenesis is probably connected to the hypoxia that characterizes inflamed tissues. Eosinophils produce VEGF and are pro-angiogenic. However, to the best of our knowledge, no study has been performed to verify the existence of a direct link between eosinophils, hypoxia and angiogenesis in allergic inflammation. METHODS: Human peripheral blood eosinophils were cultured in normoxic or hypoxic (<3% O2) conditions with or without GM-CSF (20ng/ ml). HIF-1a levels and MAPK phosphorylation were analyzed by immunoblot analysis. Angiogenic mediators release was evaluated by ELISA. RESULTS: Hypoxia increased VEGF release and HIF-1a, phosphoERK1/2 and phospho-p38 protein levels, particularly in the presence of GM-CSF. The ERK1/2 inhibitor PD98059 significantly decreased VEGF release. In the presence of GM-CSF in normoxia the inhibition decreased VEGF from 42 6 12.7 pg/ml to 22.9 6 9.97 pg/ml, and in hypoxia from 165.62 6 63.83 pg/ml to 23.77 6 6.18 pg/ml. Also with the p38 MAPK inhibitor SB203580 VEGF release decreased though not significantly. PD98059 caused HIF-1a expression decrease both in normoxia and in hypoxia, and in hypoxia it reached levels similar to the ones in normoxia. SB20358 decreased HIF-1a expression slightly but not significantly. CONCLUSIONS: Eosinophils respond to hypoxia by releasing increased amounts of pro-angiogenic factors and by up-regulating HIF-1alpha expression. This effect is augmented by the presence of GM-CSF and involves MAPK phosphorylation. These results are important for the understanding of eosinophils behaviour in inflamed tissue and for their role in angiogenesis.
189
Activation of Dendritic Cells by IL-33 Initiates a Th2 Response M. A. Rank, T. Kabayashi, K. R. Bartemes, H. Kita; Mayo Clinic, Rochester, MN. RATIONALE: IL-33, a new IL-1 family cytokine, is implicated in Th2type responses. However, the cellular targets for IL-33 are poorly understood. Because dendritic cells (DCs) are important in immune regulation, we hypothesized that DCs respond to IL-33 and trigger Th2 responses. METHODS: DCs were derived from murine bone marrow. Expression of the IL-33 receptor, ST2, was examined by FACS and real-time RT-PCR. After culturing DCs with IL-33 or LPS (as positive control) for 24 hours, activation of DCs was examined by FACS (MHC II and CD86 expression) and by ELISA (IL-6 and IL-12 production). The ability of IL-33-activated DCs to differentiate T cells was assessed by culturing DCs with naı¨ve CD4 1 T cells for 6 or 10 days and by measuring supernatant cytokines. RESULTS: ST2 mRNA was expressed abundantly in highly purified DCs, and ST2 protein was detected intracellularly by FACS. Incubation of DCs with IL-33 (as low as 1 ng/ml) increased MHC II and CD86 expression (p < 0.05) and induced IL-6 production (p < 0.01), but not IL-12. Anti-ST2 antibody inhibited IL-33-induced IL-6 production by approximately 60% (p < 0.03); LPS-induced IL-6 production was unaffected. Incubation of naive CD4 1 T cells, DCs, and IL-33 induced marked production of IL-5 and IL-13 (p < 0.05); IL-4 and IFN-g were undetectable. Incubation of DCs or naive CD4 1 T cells alone with IL-33 failed to stimulate IL-5 or IL-13 production. CONCLUSIONS: DCs respond to IL-33 through ST2. DCs plus IL-33 stimulate CD4 1 T cell production of Th2 cytokines. IL-33 and DCs may represent a new pathway to initiate Th2-type inflammation.
190
Topical N-acetyl cysteine (NAC) Reduces Late Phase Nasal Symptoms Following Ragweed Challenge C. J. Lane, D. R. Redding, K. A. Gonzalez, V. J. Cardenas, I. Boldogh, S. Sur; University of Texas Medical Branch, Galveston, TX. RATIONALE: Reactive oxygen species (ROS) generated by pollen NADPH oxidase augment aeroallergen induced airway inflammation. We examined the ability of the antioxidant NAC to modulate nasal symptoms following allergen challenge. METHODS: Ragweed sensitive patients were identified using skin prick testing and underwent nasal provocation challenges with either saline or ragweed extract (RWE.) Nasal symptom scores were recorded at baseline and at regular intervals following challenge. A late phase allergic response to RWE was defined as a difference of greater than or equal to 4 between the combined nasal symptom scores at 5 and 6 hours between RWE and saline challenge. Patients who demonstrated a late phase response to RWE then underwent challenge with RWE preceded by topical delivery of NAC in the nose. RESULTS: 3 of 8 subjects (38%) demonstrated a late phase reaction after RWE challenge and entered the open label NAC phase of the study. The total nasal symptom score (measured as change from baseline, mean 6 SEM) at 0.5 hours after allergen challenge was -1 6 0.6 for saline, 5.3 6 0.9 for RWE and 3.3 6 0.9 for NAC 1 RWE (p 5 NS vs RWE.) Scores at 5 hours were -1.3 6 0.9 for saline, 2.7 6 0.9 for RWE and -1 6 0.6 for NAC 1 RWE (p < 0.05 vs RWE.) Scores at 6 hours were -1.3 6 0.9 for saline, 2.7 6 0.9 for RWE and -1 6 1 for NAC 1 RWE (p < 0.05 vs RWE.) CONCLUSIONS: Topical NAC administered prior to RWE challenge reduced the late phase allergic response mediated nasal symptoms. Oxidative stress induced by RWE contributes to the nasal late phase response and may represent a therapeutic target for allergic airway disease.
191
Comparison of Exhaled Breath Condensate and Sputum Induced by Hyperosmotic Saline Inhalation as Indicators of Airway Inflammation Associated with Bronchial Asthma T. Shimoda1, Y. Obase2, M. Imaoka1, R. Kishikawa1, S. Nishima1; 1Clinical Research Center, Fukuoka National Hospital, Fukuoka, Japan, 2Division of Respiratory Disease, Kawasaki Medical School, Kurashiki, Japan. RATIONALE: Testing of sputum induced by hypertonic saline inhalation has been established as a useful indicator of airway inflammation associated with bronchial asthma. Although exhaled breath condensate(EBC) can be collected simply and noninvasively, it is unclear whether it can serve as a valid indicator of airway inflammation. METHODS: The study involved 291 asthmatic patients who had not undergone steroid inhalation. First, each subject breathed at rest for 15 minutes using an ECoScreen (Jaeger, Germany), and EBC was collected. Then, each subject inhaled 3% hypertonic saline for 20 minutes, and sputum was collected, centrifuged, and its supernatant stored at -808C. Both samples from each subject were subsequently assayed for 20 substances by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: Assay of supernatant of induced sputum revealed the following substances: ECP (93.2 6 84.9ng/ml), TNF-a (62.5 6 96.5 pg/ml), IL5 (3.9 6 7.5 pg/ml), VEGF (227.9 6 243.5 pg/ml), MMP-9 (66.1 6 116.7ng/ml), IL-10 (31.4 6 26.2 pg/ml), IL-12 (27.0 6 30.2 pg/ml), IL13 (13.3 6 13.8 pg/ml), Histamine (16.1 6 21.4 nM/L), Fibronectin (1.04 6 1.19 mg/ml), LTE4 (691.7 6 367.1 pg/ml), and LTB4 (161.8 6 170.1 pg/ml). The levels of TGF-b, TIMP-1, IFN-g, GM-CSF, PDGF, FGF, IL-4 and Eotaxin were beneath the limits of detection. Assay of EBC revealed two substances, IL-13 (2.9 6 2.1 pg/ml) and LTE4 (175.7 6 221.6 pg/ml), while the other substances were beneath the limits of detection. CONCLUSIONS: Induced sputum is useful as an indicator of airway inflammation associated with bronchial asthma, since both the cellular component fraction and the supernatant of sputum can be evaluated. EBC, on the other hand, contains fewer quantifiable substances. The clinical usefulness of EBC thus appears to be quite limited.
SATURDAY
Abstracts S53
J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 2