Differential Exposure of Eosinophils to Cytokines Leads to an

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protocol was shown to produce more robust symptom scores than a cumulative ... concentration. Nasal lavage samples were collected at baseline, 1 hour.

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J ALLERGY CLIN IMMUNOL VOLUME 135, NUMBER 2

Nasal Allergen Challenge (NAC) Induced Eosinophilia - the Allergic Rhinitis Clinical Investigator Collaborative (AR-CIC) Jenny Thiele, MSc1,2, Daniel Adams, BSc1, Mena Soliman, MBChB1,2, Lisa Steacy, BSc1, Anne Ellis, MD, MSc, FAAAAI1,2; 1Allergy Research Unit, Kingston General Hospital, Kingston, ON, Canada, 2Departments of Medicine and Biomedical & Molecular Science, Queen’s University, Kingston, ON, Canada. RATIONALE: The Allergic Rhinitis Clinical Investigator Collaborative (AR-CIC) is a Canadian initiative with the goal of performing standardized nasal allergen challenge (NAC) to study the effects of therapeutic agents for allergic rhinitis (AR), while allowing the identification of mechanisms and biomarkers of AR. Various NAC protocols have been described previously. The multiple cumulative allergen concentration (MCAC) NAC protocol was shown to produce more robust symptom scores than a cumulative allergen concentration (CAC) protocol. Here we examined NAC-induced eosinophilia for these two protocols. METHODS: 17 atopic and 12 non-atopic participants were enrolled for this study. Atopic individuals presented with AR symptoms in ragweed season and a supportive skin test response. During screening incremental concentrations of ragweed allergen were administered until each participant reached the qualifying symptom cut-off. For the subsequent NAC one week later, ten atopics were challenged with one dose of allergen equivalent to the cumulative amount of allergen each received during screening (CAC). Seven atopics received the cumulative of all preceding allergen doses to the qualifying concentration (QC), followed by the QC 15 minutes later (MCAC). Non-atopics were challenged with a 1:2 ragweed concentration. Nasal lavage samples were collected at baseline, 1 hour (1H) and 6 hours (6H) post NAC to determine differential counts. RESULTS: The eosinophil fraction was significantly increased in atopics following NAC when compared to non-allergics at both 1H and 6H for the CAC-protocol but only at 6H for the MCAC-protocol. CONCLUSIONS: Even though the MCAC protocol establishes more robust symptom scores, the CAC protocol appears to produce more pronounced eosinophilia.

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Differential Exposure of Eosinophils to Cytokines Leads to an Activated Phenotype in the Airways Inducing Pulmonary IL-13 Responses That Are Eosinophil Dependent Elizabeth A. Jacobsen, PhD, Dana C. Colbert, Katie R. Zellner, Cheryl A. Protheroe, William E. LeSuer, Nancy A. Lee, PhD, James J. Lee, PhD; Mayo Clinic Arizona, Scottsdale, AZ. RATIONALE: Allergic respiratory inflammation is characterized by immune responses and induced pathologies linked with increased airway IL-13. Mice deficient (CCR3-/-, IL-5-/-) or devoid (PHIL, DdblGATA-1, IL-5-/-eotaxin-1-/-) of eosinophils display reduced pulmonary IL-13 in various mouse models of allergic respiratory inflammation. Moreover, previous studies have demonstrated that eosinophils are capable of both expressing IL-13 and promoting its expression by other resident lung cells. METHODS: Purified blood-derived wild type or cytokine deficient (IL4-/- and IL-13-/-) eosinophils were either untreated or cytokine pre-treated prior to adoptive transfer into the lungs of allergen challenged mice deficient (IL-5-/-) or devoid (PHIL) of eosinophils. Allergen-induced pulmonary changes in Th2 cytokine levels, histopathologies, and accumulation of leukocyte populations were assessed. RESULTS: Eosinophils with activation-state unique activities were achieved by differential cytokine pre-treatment prior to adoptive transfer into the lungs of allergen-treated IL-5-/- or PHIL mice. GM-CSF treated eosinophils activated T cells and DCs in the lung draining lymph nodes, yet did not induce either T cell recruitment to the lung or evidence of pulmonary inflammation. In contrast, eosinophils pre-treated with both IL-33 and GM-CSF were sufficient to restore pulmonary T cell accumulation as well as induced allergen-mediated pathologies. We demonstrated in both IL-5-/- and PHILmice that the Th2 pulmonary immune responses and induced pathologies mediated by differentially activated eosinophils

were specifically dependent on the expression of IL-13 (but not IL-4) by eosinophils. CONCLUSIONS: Our data demonstrates an underappreciated and significant role for eosinophil-derived IL-13 in inducing Th2 pulmonary pathologies in mouse models of allergic respiratory inflammation.

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Preferential Production of TNFa, Compared to IL-4 and IFNg, By Differentiating Eosinophil-Basophil Cord Blood Progenitors Pia Reece, PhD1, Claudia C. K. Hui, PhD2, Judah A. Denburg, MD, FRCPC, FAAAAI1; 1Division of Clinical Immunology and Allergy, Department of Medicine, McMaster University, Hamilton, ON, Canada, 2 Division of Clinical Immunology & Allergy, McMaster University, Hamilton, ON, Canada. RATIONALE: Eosinophils are multi-functional leukocytes involved in a number of infectious and inflammatory processes, including allergic diseases. Classical TH1 (IFNg) and TH2 (IL-4) cytokines negatively impact eosinophil differentiation from cord blood (CB) in vitro. Since we have recently shown that CB progenitor cells produce cytokines that auto-regulate their own differentiation, and we have recently uncovered a novel role for TNFa positively impacting eosinophil-basophil (Eo/B) colony formation, we investigated the secretion of TNFa relative to these classical TH1 and TH2 cytokines during eosinophil differentiation from cord blood. METHODS: Fresh CB progenitor cells were stimulated with GM-CSF, IL-5 and IL-3 for 14 days in liquid media to induce eosinophil production. Cells were isolated at days 3, 7 and 10 and cytokine levels of IL-4, TNFa, IFNg, were evaluated using luminex technologies. Morphological analysis was performed to determine the presence of eosinophils in culture after 14 days. RESULTS: As compared the TH1 cytokine IFNg, TNFa was secreted at significantly greater amounts at day 3 (p50.033), day 7 (p50.040), and day 10 (p50.040) of culture. When compared to the TH2 cytokine IL-4, TNFa was secreted at significantly greater amounts at day 3 (p50.014), day 7 (p50.013), and day 10 (p50.019) of culture. CONCLUSIONS: We show for the first time that CB Eo/B progenitors preferentially secrete TNFa, compared to classic TH1 and TH2 cytokines, during their own differentiation process. Our work provides novel insights into ‘cytokine signatures’ utilized by CB progenitors that may enhance eosinophilic-basophilic inflammation in early life.

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