Experiments are proposed to examine the effects of cytokines interleukin-3 (IL-3), IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the synthetic-induction of bioactive eosinophil proteins during poiesis. Studies of the expression of m-RNA encoding the core major basic protein (MBP) and matrix proteins [eosinophil cationic protein (ECP), eosinophil- derived neurotoxin (EDN) and eosinophil peroxidase (EPO)] during eosinopoiesis will be performed using a newly developed preparation of cultured eosinophils derived from human umbilical cord blood. Data from fully mature cells will be compared to cells extracted from the peripheral blood of normal human volunteers. Studies will be performed in a newly developed guinea pig """"""""living explant"""""""" preparation and an isolated-perfused rat bronchial preparation in vivo to assess the biological activity of maturing eosinophils corresponding to cytokine-induced mRNA expression of the same cells in vitro. Data from mature isolated human peripheral eosinophils will be used at each step to validate the relevance of the biological activity of cultured cells. The central hypotheses of these studies are 1) IL-5 induces synthesis of granular protein mRNA and that incremental synthesis caused by IL-3 and GM-CSF depends upon co-stimulation with Il-5. 2) The biological activity of eosinophils will be increased if cytokine-stimulation exceeds that required for maturation alone. Cells isolated immediately postpartum from neonatal cord blood will be cultured and induced to differentiate as eosinophil progenitors by stimulation with IL-3, IL-5 and GM-CSF both singly and in combination. The mRNA expression for MBP, ECP, EDN, and EPO will be assessed in cultured cells initially and during weeks 1 (< 5% explicit differentiation), 2, 3, 4, and 5 (~ 98% purity). Using newly developed in situ methods for assessing force generation in airways activated by inflammatory stimulation, biological activity of cultured cells from each stage will be assessed as change in isometric force generation (guinea pig explant preparation) and endothelially activated increase in lung resistance (rat bronchial preparation). Activation of eosinophils in these studies will be assessed in vitro by a newly developed kinetic assay for EPO and by measurement of LTC4 secretion. All essential feasibility studies have been completed for both stages of the proposal. These studies will provide a direct correlation between the effects of cytokine-stimulation and development of biologically active proteins in eosinopoiesis. Data derived from these studies should provide insight into the mechanism by which eosinophils elicit airway hyperresponsiveness in conditions such as human allergy and asthma.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI032654-03
Application #
2067562
Study Section
Toxicology Subcommittee 2 (TOX)
Project Start
1992-04-01
Project End
1996-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Chicago
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637
Hamann, K J; Vieira, J E; Halayko, A J et al. (2000) Fas cross-linking induces apoptosis in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 278:L618-24
Munoz, N M; Hamann, K J; Rabe, K F et al. (1999) Augmentation of eosinophil degranulation and LTC(4) secretion by integrin-mediated endothelial cell adhesion. Am J Physiol 277:L802-10
White, S R; Dorscheid, D R; Rabe, K F et al. (1999) Role of very late adhesion integrins in mediating repair of human airway epithelial cell monolayers after mechanical injury. Am J Respir Cell Mol Biol 20:787-96
Hamann, K J; Dorscheid, D R; Ko, F D et al. (1998) Expression of Fas (CD95) and FasL (CD95L) in human airway epithelium. Am J Respir Cell Mol Biol 19:537-42
Hamann, K J; Neeley, S P; Dowling, T L et al. (1996) Effect of interleukin-5 exposure during in vitro eosinophilopiesis on MAC-1 adhesion molecule expression and function on cultured human eosinophils. Blood 88:3575-82
Hamann, K J; Dowling, T L; Neeley, S P et al. (1995) Hyaluronic acid enhances cell proliferation during eosinopoiesis through the CD44 surface antigen. J Immunol 154:4073-80
Neeley, S P; Hamann, K J; Dowling, T L et al. (1994) Augmentation of stimulated eosinophil degranulation by VLA-4 (CD49d)-mediated adhesion to fibronectin. Am J Respir Cell Mol Biol 11:206-13
Neeley, S P; Hamann, K J; White, S R et al. (1993) Selective regulation of expression of surface adhesion molecules Mac-1, L-selectin, and VLA-4 on human eosinophils and neutrophils. Am J Respir Cell Mol Biol 8:633-9
Hamann, K J; Strek, M E; Baranowski, S L et al. (1993) Effects of activated eosinophils cultured from human umbilical cord blood on guinea pig trachealis. Am J Physiol 265:L301-7